A rapid pupillometry protocol for clinical use: Effect of age and test-retest repeatability

A rapid pupillometry protocol for clinical use: Effect of age and test-retest repeatability | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A rapid pupillometry protocol for clinical use: Effect of age and test-retest repeatability Jason C. Park, J. Jason McAnany This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7383219/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Nov, 2025 Read the published version in Documenta Ophthalmologica → Version 1 posted 9 You are reading this latest preprint version Abstract Purpose: Pupillometry is most commonly performed in laboratory settings using specialized, non-portable instruments that require lengthy test protocols. The purpose of this study was to develop and evaluate a rapid, clinically-applicable pupillometry protocol using a commercially available, portable, handheld instrument. Methods: Thirty-seven healthy individuals (ages 21- 61 years) participated in three experiments. In each experiment, the pupillary light reflex (PLR) was elicited by full-field, 500-ms chromatic flashes (470 nm and 621 nm; 12,000 Td). Experiment I evaluated the minimum dark adaptation (DA) time needed to achieve maximum PLRs. Experiment II determined the effect of age. Experiment III estimated PLR test-retest repeatability. For all experiments, baseline pupil size (BL; 1 sec before flash onset), maximum pupil constriction (MPC) following the flash, and post-illumination pupillary response (PIPR; median size 6 - 8 sec after flash offset) were quantified. Results: Experiment I showed that from 1 - 3 min of DA, BL and MPC increased slightly (0.27 mm and 5%, respectively), whereas the PIPR increased considerably (17%). The responses did not change appreciably after 3 min, therefore a 3-min DA period was used for Experiments II and III. Experiment II showed a trend for BL and MPC to decrease with age, but correlations with age were not statistically significant (all p > 0.05). PIPR was independent of age (r = -0.01; p = 0.96). Experiment III showed test-retest repeatability of approximately 1 mm for BL, and 10% for MPC and PIPR, indicating good repeatability. Conclusion: The proposed approach is useful for measuring the MPC and PIPR across a broad range of ages and baseline pupil sizes. Given the device portability and short test duration (approximately 5 minutes including DA), this approach has promising clinical utility. Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Pupil size is adaptively modulated by the iris sphincter and dilator muscles to control the amount of light entering the eye. Although several factors affect pupil size, light stimulation is the primary driver and the response of the pupil to changes in illumination has been termed the pupillary light reflex (PLR). The PLR is largely mediated by intrinsically photosensitive retinal ganglion cells (ipRGCs) that also receive inputs from rod and cone photoreceptors [ 1 – 3 ]. Given that the PLR is dependent on the function of outer- and inner-retinal neurons, as well as afferent and efferent post-retina pathways, the PLR has become of interest in Ophthalmology and Neurology. Pupillometric assessments have been reported in patients with acquired [ 4 – 10 ] and inherited [ 11 – 16 ] ocular dysfunction. Beyond the retina, pupillometry has been applied to study central nervous system function in conditions including traumatic brain injury [ 17 – 19 ], Alzheimer’s disease [ 20 – 24 ], and Parkinson’s disease [ 25 – 29 ]. Traditionally, clinical assessment of the pupil has been performed qualitatively using a penlight (e.g., static measurement of pupil diameter under light-adapted conditions or with the “swinging flashlight test” to detect relative afferent pupillary defects). In recent decades, however, commercially available infrared videography systems have been developed that can provide quantitative measures of pupil size and reactivity. These infrared systems permit assessing pupil dynamics under both light- and dark-adapted conditions, which has become of interest in the clinical assessment of patients who have cone and rod photoreceptor dystrophies [ 12 , 13 , 30 , 31 ]. The PLR elicited by a high-luminance, short-wavelength stimulus has been of particular interest, as this combination allows assessment of inner retinal function by stimulating ipRGCs. Specifically, the PLR elicited by a brief (≤ 1 s), high luminance, short-wavelength stimulus is characterized by a rapid, transient constriction that is thought to be rod- and cone-mediated [ 32 – 35 ]. The transient constriction is followed by a slow, sustained constriction that is often referred to as the post-illumination pupil response (PIPR) and is believed to be generated by melanopsin activation within ipRGCs [ 32 , 34 , 36 ]. Despite the promise of clinical pupillometry, the technique has largely been restricted to research applications in academic medical centers. Wider application has, in large part, been hampered by instrument cost, portability, and burdensome test lengths. There have been attempts to create portable pupillometers, including implementation using smart phones [ 37 , 38 ] and other devices [ 39 – 42 ], but none are used routinely in clinical practice. These portable pupillometry devices differ considerably in their ease of use, data analysis capabilities, and ability to elicit PLRs mediated by the rod-, cone-, and melanopsin-pathways. Recently, a portable, hand-held instrument for retinal electrophysiology (RETeval, LKC Technologies Inc., Gaithersburg, MD, USA) has been adapted to measure the PLR [ 16 , 43 , 44 ]. The instrument can produce stimuli that are high luminance and nearly full field in size, which are important features for eliciting a robust PIPR. The first report with this device [ 43 ] elicited pupil responses using low (50 cd/m 2 ) and high (316 cd/m 2 ) luminance flashes of blue light (470 nm) and the pupil size was recorded using built-in software, allowing for quantitative analyses. This protocol used only 3 minutes of dark-adaptation (DA), which expedited testing. Data from this small clinical study indicated that the maximum transient constriction differed between healthy control subjects (N = 5) and patients with optic neuropathy (N = 10); the PIPR was not reported. This report was followed by a second study [ 44 ] that obtained pupil responses from 32 healthy, young subjects (20–24 years of age) using 100 cd/m 2 red and blue flashes of light. The DA period was 10 minutes; baseline pupil size and maximum transient constriction were reported. Robust pupil responses were elicited with the RETeval and the responses were in excellent agreement with a lab-based pupillometer (Iriscorder Dual, Hamamatsu Photonics, Hamamatsu, Japan). Most recently, the RETeval was used to examine pupil responses in patients with early onset high myopia (N = 14) and heathy emmetropic controls (N = 12) [ 16 ]. Following 10 min of DA, 250 cd/m 2 red and blue flashes were presented and the baseline, maximum transient constriction, and PIPR were recorded. Reduced PIPRs were observed in myopic subjects who had MYP-26 gene variants. All three studies show promise for the clinical application of portable pupillometry with the RETeval. However, the study protocols tended to be lengthy, data were obtained primarily from young subjects, and measures of test-retest repeatability were not performed. An additional limitation is that these studies used a constant flash luminance to elicit pupil constrictions, regardless of the subject’s baseline pupil size. This results in different amounts of light entering the eye, which may contribute to the inter-subject variability in these previous reports. Additionally, the studies were focused on the maximum transient constriction following the flash, with only one study reporting the PIPR. Thus, the purpose of the present study was to build on these previous reports to develop a rapid, clinically-applicable chromatic pupillometry protocol. In contrast to previous studies, stimuli were defined in Trolands, which accounted for differences in baseline pupil sizes among individuals, ensuring the same amount of light entered the eye of each subject. In Experiment I, the effect of DA duration was studied to define the minimum time needed to achieve maximum responses. In Experiment II, the effects of age on the transient and PIPR components of the PLR were determined. In Experiment III, test-retest repeatability of the transient and PIPR components was studied. METHODS Subjects Thirty-seven healthy subjects were recruited from the University of Illinois Chicago, Department of Ophthalmology and Visual Sciences. The subjects were 20 to 61 years old (average 42 ± 13; 20 females). Subjects were excluded if there was a history of, or if they presented with, diabetes, glaucoma, optic neuropathy, or any other ophthalmic condition known to affect retinal or pupil function. Additionally, subjects with asymmetrically shaped pupils were excluded and no subject had a history of a neurologic event (cerebrovascular stroke or transient ischemic attack). The research followed the tenets of the Declaration of Helsinki and was approved by an institutional review board of the University of Illinois Chicago. All subjects provided written informed consent. Apparatus, stimuli, and procedure Pupillometry was performed on the right eye of each subject using the RETeval device. The stimuli consisted of red (621 nm) and blue (470 nm) LED-generated flashes. The flash duration was 500 milliseconds and a constant stimulus flash retinal illuminance (12,000 Td) was generated by monitoring the subject’s pupil size using a built-in IR-sensitive camera and adjusting luminance accordingly. The IR camera has 0.08 mm/pixel resolution at the iris and a 28.3 Hz recording rate. Pupil diameters were exported using an extraction tool supplied by the manufacturer. The waveforms were processed using a custom script programmed in MATLAB R2021b (MathWorks Inc., Natick, MA), which allowed for semi-automated analysis as described elsewhere [ 7 , 32 ]. For ease of processing, data were extracted and analyzed, but the output of the pupil metrics recorded in this study can now be obtained directly from the device’s display or from automatically generated accompanying PDF reports. The automatically generated reports were not available at the time the study was initiated, which necessitated the use of the off-line semi-automated approach. Subsequent comparison of the semi-automated analysis with the manufacturer’s automatically processed values found excellent consistency. Measurement of the PLR elicited by the red flash always preceded the blue flash with an inter-stimulus interval (ISI) of at least 30 sec. Typically, a single PLR was obtained, but in cases of blinks or noise, the PLR measurement was repeated with an ISI of at least 60 sec. Baseline pupil size (BL) was defined as the median pupil size (mm) during the 1-sec preceding each stimulus onset, and each PLR sweep was normalized by the BL. Maximum pupil constriction (MPC) was defined as the difference between BL and the minimum pupil size after stimulus onset (normalized units). The post-illumination pupil response (PIPR) was defined as difference between the BL and the median pupil size at 6–8 sec after stimulus offset (normalized units). The effect of DA duration was examined in Exp I in 13 subjects (44 ± 13.1 years; 7 female). For this experiment, PLRs were only elicited with the blue stimulus. The subjects were dark-adapted for randomly assigned duration of 1, 2, 3, or 4 min, then exposed to the blue flash. After recording, the subject was exposed to room light for least 2 min before the next randomly selected DA period began. The effect of age on BL, MPC, and PIPR was evaluated in Exp II in 37 subjects. Exp III examined test-retest repeatability using Bland-Altman analysis. A subset of 28 subjects (41.8 ± 11.3 years; 15 females) was tested twice using the same protocol developed in Exp II. The interval between two tests ranged from approximately 15 minutes to 292 days (mean test interval of 53.7 ± 105.8 days). RESULTS Figure 1 a shows the mean normalized pupil traces following 1 min (black), 2 min (red), 3 min (blue), or 4 min (green) of DA. The PLRs were characterized by an initial transient constriction (“MPC;” rod- and cone-mediated) followed by a sustained constriction that persisted for several seconds after stimulus offset (“PIPR;” melanopsin-mediated). It is clear that both the MPC and PIPR increased from 1 to 3 min of DA, with relatively little change from 3 to 4 min. Figure 1 b plots the mean (± SEM) MPC (red) and PIPR (blue) amplitudes as a function of DA duration. A two-parameter exponential growth to maximum function provide a good description of the data (solid lines). The MPC increased in amplitude from 1 to 2 min of DA and was essentially constant thereafter. The PIPR increased in amplitude from 1 to 3 min of DA, and was essentially constant thereafter. Of note, there was a clear difference in MPC and PIPR amplitude following 1 min DA, a small difference following 2 min DA, and little difference following 3 or 4 min DA. Similar values of MPC and PIPR are indicative of minimal return to baseline at 6–8 sec following the flash due to a robust melanopsin-mediated PIPR (apparent in the Fig. 1 a). Based on these results, a 3-min DA period was used for Exp II and III. Figure 2 plots BL (a), MPC (b), and PIPR (c) as a function of the subject’s age; data are shown for red and blue stimuli and are fit with linear regression lines (dashed). BLs prior to the red and blue flashes were nearly identical for a given subject, with the red and blue regression lines essentially overlapping (Fig. 2 a). There was a trend for BL to decrease with increasing age (red and blue MPC r >-0.23, p > 0.16), but this was not statistically significant. Figure 2 b shows that the MPC elicited by the blue stimulus was larger than that elicited by the red stimulus. There was a trend for the blue MPC to decrease with increasing age (r = -0.32, p = 0.054), but this was not statistically significant; red MPC was independent of age (r = 0.003, p = 0.99). Figure 2 c shows that the PIPR elicited by the red stimulus was negligible (mean red PIPR = 0.06) and there was no significant correlation with age (r = 0.26, p = 0.12). The blue stimulus elicited a large PIPR (mean blue PIPR = 0.46), but there was no significant correlation with age (r = -0.01, p = 0.96). Previous work identified weak effects of sex on the PLR, with females having slightly larger MPC under dark-adapted conditions, compared to males [ 45 , 46 ]. A possible effect of sex on BL, MPC, and PIPR was evaluated in the present data set. Two-way ANOVAs were conducted with stimulus wavelength and subject sex as main effects. ANOVA indicated no significant effect of sex on BL, MPC, or PIPR (F 0.43) and no interaction between sex and stimulus wavelength for any measure (F 0.07). Figure 3 plots the difference in BL for the two repeat tests as a function of the mean of the two tests in the form of a Bland-Altman plot. Data are only shown for the BL preceding the red stimulus. The mean BL difference for the two tests was 0.04 mm (0.7%; solid blue line) and the upper and lower limits of repeatability (± 95%; dashed lines) were + 0.98 mm and − 0.90 mm, respectively. The difference between the two tests was not significantly correlated with the overall BL size (r = 0.01, p = 0.96). Figure 4 a and 4 b represent repeat measurements of the red and blue MPC in the same Bland-Altman format as Fig. 3 . Figure 4 a shows the mean difference between tests for the red MPC was 0.01, with an upper limit of repeatability of 0.10 and a lower limit of -0.08. Figure 4 b shows the mean difference between tests for the blue MPC was 0.01, with an upper limit of repeatability of 0.10 and a lower limit of -0.09. Figure 4 c shows the mean difference between the PIPR measurements for the blue stimulus. The mean difference between tests was − 0.001, with upper and lower limits of repeatability of + 0.11 and − 0.11, respectively. The difference between the two tests was not significantly correlated with the overall MPC or PIPR size (all p > 0.37) DISCUSSION The purpose of this study was to develop and evaluate a rapid, clinically-applicable pupillometry protocol using a commercially available, portable, handheld instrument. The primary findings were 1) a 3-min DA period is sufficient to elicit robust MPCs and PIPRs; 2) there is no statistically significant relationship between age and BL, MPC, or PIPR in our sample of subjects across the ages of 26–61 years; 3) test-retest repeatability was good, with differences within approximately 10% observed. The total test time, including DA, for one eye was approximately 5 min. DA periods of 10 to 15 min are commonly employed in pupillometry studies [ 32 , 34 , 47 ]. Although less demanding than the ISCEV standard 20 min DA for full-field electroretinography [ 48 ], DA remains a burden in clinical testing. Data of the present study show that 3 min is sufficient to elicit large MPCs and PIPRs in healthy, visually-normal individuals. This finding is consistent with the conclusion of Bindiganavale [ 43 ] who reported maximum pupillary constrictions following three minutes of DA. We observed minimal differences in pupil response between 3 and 4 min, and therefore used 3 min DA to expedite testing. Although the stimulus parameters selected for this study elicited large MPCs and PIPRs after only 3 min of DA, a 3-min DA period may not elicit maximum MPCs and PIPRs for other stimuli. For example, Wang et al. [ 47 ] reported that the PIPR increased over a time-course of approximately 20 min of DA for a 100 cd/m 2 flash (1 sec; 465 nm). It is also important to note that that present findings were obtained only from healthy, visually-normal subjects and that DA dynamics may differ in patient populations. Somewhat surprisingly, there was relatively little effect of age on the pupil metrics that were assessed in the present study. BL decreased by 0.17 mm with each increasing decade of age, but this relationship did not achieve statistical significance. “Senile miosis” is a well-known effect of aging [ 49 – 52 ]. It is possible that the effect of age on BL may have been more apparent in the present dataset if a larger number of subjects older than 60 years of age were included. Studies reporting the effect of age on the human PIPR have been inconsistent [ 53 – 55 ]. Age-related loss of ipRGC subtypes has been reported in the human retina [ 56 ], but the loss appears most prominently after the age of 70. Rather than loss of ipRGCs, reduced blue light sensitivity due to cataract formation may be a more influential factor to consider when interpreting chromatic pupillometry results across age. Test-retest repeatability in the present sample of subjects was good, with differences less than approximately 10% observed, defined by Bland-Altman analyses. Herbst et al. [ 57 ] reported intraclass coefficients (ICCs) for maximum constrictions elicited by red and blue flashes measured with a custom pupilometer to be 0.7 to 0.8, which is similar to our MPC ICC value of 0.71. Li et al. [ 58 ] reported ICCs of 0.84 to 0.94 for the PIPR elicited by full-field blue flashes, which is similar to our PIPR ICC of 0.97. Of note, the present study is underpowered to assess test-retest repeatability within a narrow confidence interval. To define repeatability within a 10% confidence interval, for example, 190 subjects would be needed. Our sample size of 27 subjects provides a much larger confidence interval of 27%. Nevertheless, the data provided herein represents a first step toward a potential standardized approach to rapid portable pupillometry. In summary, we provide evidence that a short-duration, clinically applicable, chromatic pupillometry test can be used to assess function in healthy individuals. The test is administered quickly (approximately 5 min for one eye), is well tolerated, and repeatable. Declarations AUTHOR CONTRIBUTIONS Both authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by JCP. The first draft of the manuscript was written by JJM and JCP. Both authors read and approved the final manuscript. CONFLICTS OF INTEREST The authors report no conflicts of interest. The research was supported by the National Institutes of Health research grants R01EY026004 (JM), P30EY001792 (core grant), an unrestricted departmental grant from Research to Prevent Blindness. Compliance with Ethical Standards The studies were performed in accordance with the tenants of the Declaration of Helsinki, institutional review board approval was obtained at the University of Illinois at Chicago, and the experiments were undertaken with the understanding and written consent of each subject. Statement on the welfare of animals No animal experiments were performed. Informed consent The subjects consented to participate in the study. Grant Information: National Institutes of Health research grants R01EY026004 (JM), P30EY001792 (core grant), an unrestricted departmental grant from Research to Prevent Blindness. References Spitschan M (2019) Photoreceptor inputs to pupil control. J Vis 19(9):5 Guler AD, Ecker JL, Lall GS, Haq S, Altimus CM, Liao HW, Barnard AR, Cahill H, Badea TC, Zhao H, Hankins MW, Berson DM, Lucas RJ, Yau KW, Hattar S (2008) Melanopsin cells are the principal conduits for rod-cone input to non-image-forming vision. Nature 453(7191):102–105 Hall CA, Chilcott RP (2018) Eyeing up the Future of the Pupillary Light Reflex in Neurodiagnostics. Diagnostics 8 (1) Feigl B, Zele AJ, Fader SM, Howes AN, Hughes CE, Jones KA, Jones R (2012) The post-illumination pupil response of melanopsin-expressing intrinsically photosensitive retinal ganglion cells in diabetes. Acta Ophthalmol 90(3):e230–234 Ferrari GL, Marques JL, Gandhi RA, Heller SR, Schneider FK, Tesfaye S, Gamba HR (2010) Using dynamic pupillometry as a simple screening tool to detect autonomic neuropathy in patients with diabetes: a pilot study. Biomed Eng Online 9:26 Karavanaki K, Davies AG, Hunt LP, Morgan MH, Baum JD (1994) Pupil size in diabetes. Arch Dis Child 71(6):511–515 Park JC, Chen YF, Blair NP, Chau FY, Lim JI, Leiderman YI, Shahidi M, McAnany JJ (2017) Pupillary responses in non-proliferative diabetic retinopathy. Sci Rep 7:44987 Park JC, Moss HE, McAnany JJ (2016) The Pupillary Light Reflex in Idiopathic Intracranial Hypertension. Invest Ophthalmol Vis Sci 57(1):23–29 Najjar RP, Sharma S, Atalay E, Rukmini AV, Sun C, Lock JZ, Baskaran M, Perera SA, Husain R, Lamoureux E, Gooley JJ, Aung T, Milea D (2018) Pupillary Responses to Full-Field Chromatic Stimuli Are Reduced in Patients with Early-Stage Primary Open-Angle Glaucoma. Ophthalmology 125(9):1362–1371 Kelbsch C, Maeda F, Strasser T, Blumenstock G, Wilhelm B, Wilhelm H, Peters T (2016) Pupillary responses driven by ipRGCs and classical photoreceptors are impaired in glaucoma. Graefes Arch Clin Exp Ophthalmol 254(7):1361–1370 Kawasaki A, Crippa SV, Kardon R, Leon L, Hamel C (2012) Characterization of pupil responses to blue and red light stimuli in autosomal dominant retinitis pigmentosa due to NR2E3 mutation. Invest Ophthalmol Vis Sci 53(9):5562–5569 Collison FT, Park JC, Fishman GA, McAnany JJ, Stone EM (2015) Full-Field Pupillary Light Responses, Luminance Thresholds, and Light Discomfort Thresholds in CEP290 Leber Congenital Amaurosis Patients. Invest Ophthalmol Vis Sci 56(12):7130–7136 Collison FT, Park JC, Fishman GA, Stone EM, McAnany JJ (2016) Two-color pupillometry in enhanced S-cone syndrome caused by NR2E3 mutations. Doc Ophthalmol 132(3):157–166 Kawasaki A, Collomb S, Leon L, Munch M (2014) Pupil responses derived from outer and inner retinal photoreception are normal in patients with hereditary optic neuropathy. Exp Eye Res 120:161–166 Hyde RA, Kratunova E, Park JC, McAnany JJ (2022) Cone pathway dysfunction in Jalili syndrome due to a novel familial variant of CNNM4 revealed by pupillometry and electrophysiologic investigations. Ophthalmic Genet 43(2):268–276 Barboni MTS, Szell N, Sohajda Z, Feher T (2024) Pupillary Light Reflex Reveals Melanopsin System Alteration in the Background of Myopia-26, the Female Limited Form of Early-Onset High Myopia. Invest Ophthalmol Vis Sci 65(8):6 The Brain Trauma Foundation. The American Association of Neurological Surgeons (2000) The Joint Section on Neurotrauma and Critical Care. Pupillary diameter and light reflex. J Neurotrauma 17(6–7):583–590 Yuhas PT, Shorter PD, McDaniel CE, Earley MJ, Hartwick AT (2017) Blue and Red Light-Evoked Pupil Responses in Photophobic Subjects with TBI. Optometry Vis science: official publication Am Acad Optometry 94(1):108–117 Mostafa J, Porter J, Queener HM, Ostrin LA (2021) Intrinsically photosensitive retinal ganglion cell-driven pupil responses in patients with traumatic brain injury. Vis Res 188:174–183 Frost S, Kanagasingam Y, Sohrabi H, Bourgeat P, Villemagne V, Rowe CC, Macaulay SL, Szoeke C, Ellis KA, Ames D, Masters CL, Rainey-Smith S, Martins RN, Group AR (2013) Pupil response biomarkers for early detection and monitoring of Alzheimer's disease. Curr Alzheimer Res 10(9):931–939 Tales A, Troscianko T, Lush D, Haworth J, Wilcock GK, Butler SR (2001) The pupillary light reflex in aging and Alzheimer's disease. Aging 13(6):473–478 Oh AJ, Amore G, Sultan W, Asanad S, Park JC, Romagnoli M, La Morgia C, Karanjia R, Harrington MG, Sadun AA (2019) Pupillometry evaluation of melanopsin retinal ganglion cell function and sleep-wake activity in pre-symptomatic Alzheimer's disease. PLoS ONE 14(12):e0226197 Romagnoli M, Stanzani Maserati M, De Matteis M, Capellari S, Carbonelli M, Amore G, Cantalupo G, Zenesini C, Liguori R, Sadun AA, Carelli V, Park JC, La Morgia C (2020) Chromatic Pupillometry Findings in Alzheimer's Disease. Front NeuroSci 14:780 La Morgia C, Mitolo M, Romagnoli M, Stanzani Maserati M, Evangelisti S, De Matteis M, Capellari S, Bianchini C, Testa C, Vandewalle G, Santoro A, Carbonelli M, D'Agati P, Filardi M, Avanzini P, Barboni P, Zenesini C, Baccari F, Liguori R, Tonon C, Lodi R, Carelli V (2023) Multimodal investigation of melanopsin retinal ganglion cells in Alzheimer's disease. Ann Clin Transl Neurol 10(6):918–932 Giza E, Fotiou D, Bostantjopoulou S, Katsarou Z, Karlovasitou A (2011) Pupil light reflex in Parkinson's disease: evaluation with pupillometry. Int J Neurosci 121(1):37–43 Joyce DS, Feigl B, Kerr G, Roeder L, Zele AJ (2018) Melanopsin-mediated pupil function is impaired in Parkinson's disease. Sci Rep 8(1):7796 Feigl B, Dumpala S, Kerr GK, Zele AJ (2020) Melanopsin Cell Dysfunction is Involved in Sleep Disruption in Parkinson's Disease. J Parkinson's disease 10(4):1467–1476 Tabashum T, Zaffer A, Yousefzai R, Colletta K, Jost MB, Park Y, Chawla J, Gaynes B, Albert MV, Xiao T (2021) Detection of Parkinson's Disease Through Automated Pupil Tracking of the Post-illumination Pupillary Response. Front Med 8:645293 Gaynes BI, Zaffer A, Yousefzai R, Chazaro-Cortes M, Colletta K, Kletzel SL, Jost MB, Park Y, Chawla J, Albert MV, Xiao T (2022) Variable abnormality of the melanopsin-derived portion of the pupillary light reflex (PLR) in patients with Parkinson's disease (PD) and parkinsonism features. Neurol sciences: official J Italian Neurol Soc Italian Soc Clin Neurophysiol 43(1):349–356 Charng J, Jacobson SG, Heon E, Roman AJ, McGuigan DB 3rd, Sheplock R, Kosyk MS, Swider M, Cideciyan AV (2017) Pupillary Light Reflexes in Severe Photoreceptor Blindness Isolate the Melanopic Component of Intrinsically Photosensitive Retinal Ganglion Cells. Invest Ophthalmol Vis Sci 58(7):3215–3224 McAnany JJ, Park JC, Fishman GA, Collison FT (2020) Full-Field Electroretinography, Pupillometry, and Luminance Thresholds in X-Linked Retinoschisis. Invest Ophthalmol Vis Sci 61(6):53 Park JC, Moura AL, Raza AS, Rhee DW, Kardon RH, Hood DC (2011) Toward a clinical protocol for assessing rod, cone, and melanopsin contributions to the human pupil response. Invest Ophthalmol Vis Sci 52(9):6624–6635 Kostic C, Crippa SV, Martin C, Kardon RH, Biel M, Arsenijevic Y, Kawasaki A (2016) Determination of Rod and Cone Influence to the Early and Late Dynamic of the Pupillary Light Response. Invest Ophthalmol Vis Sci 57(6):2501–2508 Adhikari P, Zele AJ, Feigl B (2015) The Post-Illumination Pupil Response (PIPR). Invest Ophthalmol Vis Sci 56(6):3838–3849 Philibert M, Milea D (2024) Basics, benefits, and pitfalls of pupillometers assessing visual function. Eye (Lond) 38(12):2415–2421 Kardon R, Anderson SC, Damarjian TG, Grace EM, Stone E, Kawasaki A (2011) Chromatic pupillometry in patients with retinitis pigmentosa. Ophthalmology 118(2):376–381 McAnany JJ, Smith BM, Garland A, Kagen SL (2018) iPhone-based Pupillometry: A Novel Approach for Assessing the Pupillary Light Reflex. Optometry Vis science: official publication Am Acad Optometry 95(10):953–958 Tan HH, Lee KC, Chen YR, Huang YC, Ke RS, Horng GJ, Chen KT (2025) Using smartphone pupillometer application to measure pupil size and light reflex: An unsuccessful prototype and analysis of the causes of failure. Medicine 104(9):e41682 Emelifeonwu JA, Reid K, Rhodes JK, Myles L (2018) Saved by the Pupillometer! - A role for pupillometry in the acute assessment of patients with traumatic brain injuries? Brain Injury 32(5):675–677 Jehu DA, Bolgla LA, Armas S, Dutton F (2025) Assessing the Inter-Rater and Inter-Trial Reliability of the NeurOptics Pupillary Light Response-3000 Pupillometer. Int J sports Phys therapy 20(2):157–167 Najjar RP, Rukmini AV, Finkelstein MT, Nusinovici S, Mani B, Nongpiur ME, Perera S, Husain R, Aung T, Milea D (2023) Handheld chromatic pupillometry can accurately and rapidly reveal functional loss in glaucoma. Br J Ophthalmol 107(5):663–670 Kohnen T, Terzi E, Buhren J, Kohnen EM (2003) Comparison of a digital and a handheld infrared pupillometer for determining scotopic pupil diameter. J Cataract Refract Surg 29(1):112–117 Bindiganavale MP, Moss HE (2021) Development and Implementation of a Handheld Pupillometer for Detection of Optic Neuropathies. Curr Eye Res 46(9):1432–1435 Asakawa K, Imai M, Ohta M, Kawata N, Kawatsu N, Ishikawa H (2023) Pupil assessment with a new handheld pupillometer in healthy subjects. Int Ophthalmol 43(1):51–61 Ishikawa M (2021) Clinical factors affecting pupillary light reflex parameters: a single-centre, cross-sectional study. Ophthalmic Physiol Opt 41(5):952–960 Fan X, Hearne L, Lei B, Miles JH, Takahashi N, Yao G (2009) Weak gender effects on transient pupillary light reflex. Auton neuroscience: basic Clin 147(1–2):9–13 Wang B, Shen C, Zhang L, Qi L, Yao L, Chen J, Yang G, Chen T, Zhang Z (2015) Dark adaptation-induced changes in rod, cone and intrinsically photosensitive retinal ganglion cell (ipRGC) sensitivity differentially affect the pupil light response (PLR). Graefes Arch Clin Exp Ophthalmol 253(11):1997–2005 Robson AG, Frishman LJ, Grigg J, Hamilton R, Jeffrey BG, Kondo M, Li S, McCulloch DL (2022) ISCEV Standard for full-field clinical electroretinography (2022 update). Doc Ophthalmol Adv Ophthalmol 144(3):165–177 Kornzweig A (1954) Physiological effects of age on the visual process. Sight Sav Rev 24:130–138 Birren JE, Casperson RC, Botwinick J (1950) Age Changes in Pupil Size. J Gerontol 5(3):216–221 IE L (1979) Pupillary changes related to age. Topic in Neuro-Ophthalmology Robertson GW, Yudkin J (1944) Effect of age upon dark adaptation. J Physiol 103(1):1–8 Chen Y, Pinto AA, Paulsen AJ, Schubert CR, Hancock LM, Klein BE, Klein R, Cruickshanks KJ (2019) The Post-illumination Pupil Response (PIPR) Is Associated With Cognitive Function in an Epidemiologic Cohort Study. Front Neurol Volume 10–2019 Herbst K, Sander B, Lund-Andersen H, Broendsted AE, Kessel L, Hansen MS, Kawasaki A (2012) Intrinsically photosensitive retinal ganglion cell function in relation to age: a pupillometric study in humans with special reference to the age-related optic properties of the lens. BMC Ophthalmol 12:4 Klevens AM, Taylor ML, Wescott DL, Gamlin PD, Franzen PL, Hasler BP, Siegle G, Roecklein KA (2024) The role of retinal irradiance estimates in melanopsin-driven retinal responsivity: a reanalysis of the post-illumination pupil response in seasonal affective disorder. Sleep 47 (9) Esquiva G, Lax P, Perez-Santonja JJ, Garcia-Fernandez JM, Cuenca N (2017) Loss of Melanopsin-Expressing Ganglion Cell Subtypes and Dendritic Degeneration in the Aging Human Retina. Front Aging Neurosci 9:79 Herbst K, Sander B, Milea D, Lund-Andersen H, Kawasaki A (2011) Test-retest repeatability of the pupil light response to blue and red light stimuli in normal human eyes using a novel pupillometer. Front Neurol 2:10 Lei S, Goltz HC, Chandrakumar M, Wong AM (2015) Test-retest reliability of hemifield, central-field, and full-field chromatic pupillometry for assessing the function of melanopsin-containing retinal ganglion cells. Invest Ophthalmol Vis Sci 56(2):1267–1273 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 15 Nov, 2025 Read the published version in Documenta Ophthalmologica → Version 1 posted Editorial decision: Revision requested 09 Oct, 2025 Reviews received at journal 08 Oct, 2025 Reviewers agreed at journal 08 Oct, 2025 Reviews received at journal 24 Sep, 2025 Reviewers agreed at journal 16 Sep, 2025 Reviewers invited by journal 12 Sep, 2025 Editor assigned by journal 20 Aug, 2025 Submission checks completed at journal 20 Aug, 2025 First submitted to journal 15 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7383219","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":515699575,"identity":"a597dc7d-5807-4d3a-b06f-21dd55a6ff11","order_by":0,"name":"Jason C. Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACxgYQeeC/HD+EyUy0FmZjyQZitUDAAeZEgwNgFhFamNvPHvxccYYtwfhGcuMHhgrrxAaCDuvJS5Y8c4Mnz+xGYrMEw5l0IrTM4DGQbPggUQzU0sbA2HaYKC3GPxs+GCRungHS8o84LWaSDTcSEjdIgLQ0EKOlJy/NsuHMAWOJMw+bJRKOpRsT1GLYfvbwzYZjB+T429MffvhQYy1LWEsDDxIvgZByEJBn4CGsaBSMglEwCkY4AACHS0MhX4nXggAAAABJRU5ErkJggg==","orcid":"","institution":"University of Illinois Chicago","correspondingAuthor":true,"prefix":"","firstName":"Jason","middleName":"C.","lastName":"Park","suffix":""},{"id":515699576,"identity":"8f9c4a96-46cb-49fd-92bd-ee57873b3ccd","order_by":1,"name":"J. Jason McAnany","email":"","orcid":"","institution":"University of Illinois Chicago","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"Jason","lastName":"McAnany","suffix":""}],"badges":[],"createdAt":"2025-08-15 17:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7383219/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7383219/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10633-025-10068-5","type":"published","date":"2025-11-15T15:58:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91732624,"identity":"00c12195-bb4f-4d5e-90c1-43e126d51f34","added_by":"auto","created_at":"2025-09-19 16:28:21","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":289280,"visible":true,"origin":"","legend":"","description":"","filename":"DOOPPupilprotocolFinal.doc","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/5720da80dbeafc5cdb8e98d3.doc"},{"id":91732625,"identity":"ce54a413-41d9-41f4-9510-2869c390fab4","added_by":"auto","created_at":"2025-09-19 16:28:21","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":289331,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/1892f5204ab122a48fdb677c.tif"},{"id":91732854,"identity":"574dfc4e-af22-43d4-bdcd-959441927b58","added_by":"auto","created_at":"2025-09-19 16:36:21","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":312269,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/1b9ba16a86d9bc7bf2022a83.tif"},{"id":91732856,"identity":"c86a7f9c-4ea5-4613-bf24-70accb939560","added_by":"auto","created_at":"2025-09-19 16:36:21","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":244618,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/bd8708a1df596783a4fe9cb7.tif"},{"id":91732860,"identity":"90c8cd07-b576-4b0b-ba72-e7fdfb87a0cc","added_by":"auto","created_at":"2025-09-19 16:36:21","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":298757,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/dd4087e37b071e0e2a1d13b4.tif"},{"id":91732622,"identity":"fbe7e57d-e741-444b-94f2-ee37e7bfcab1","added_by":"auto","created_at":"2025-09-19 16:28:21","extension":"json","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4628,"visible":true,"origin":"","legend":"","description":"","filename":"a1213b030d174d5bb5fd07b25e758ff2.json","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/78a8fee8f64a20c9fc821a7d.json"},{"id":91732637,"identity":"1c87b3b7-e93a-4192-b7d2-86f17f8e79a3","added_by":"auto","created_at":"2025-09-19 16:28:21","extension":"xml","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":118741,"visible":true,"origin":"","legend":"","description":"","filename":"a1213b030d174d5bb5fd07b25e758ff21enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/aa6f36654196babd488aa5a9.xml"},{"id":91732627,"identity":"302007e7-0ff2-451d-9d2a-c12b85556cf3","added_by":"auto","created_at":"2025-09-19 16:28:21","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":289331,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/b2cde1cf734c85a1e36e7bf4.tif"},{"id":91732858,"identity":"f56db3cc-8217-4a9b-afe9-582116ce0a06","added_by":"auto","created_at":"2025-09-19 16:36:21","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":312269,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/9fe188d15d808e4a68fc9e2d.tif"},{"id":91732638,"identity":"c924e961-656a-431a-8dfa-7d4f86b649a6","added_by":"auto","created_at":"2025-09-19 16:28:21","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":244618,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/9aec5bb7325a12e6964fe9f8.tif"},{"id":91732862,"identity":"951680c7-c860-4a6b-962b-4a2d67bf2929","added_by":"auto","created_at":"2025-09-19 16:36:21","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":298757,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/e09be63eda2ced6a681c3f17.tif"},{"id":91733427,"identity":"b44bd9d4-2c08-4a5a-bacd-61c41dcf5ada","added_by":"auto","created_at":"2025-09-19 16:44:21","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":63808,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/207daad0ba137d304a1880d4.png"},{"id":91732634,"identity":"f257b88d-8f5f-45be-9a81-c8eed48cba69","added_by":"auto","created_at":"2025-09-19 16:28:21","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98799,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/8bd3405a22111381a1d8d2f1.png"},{"id":91732855,"identity":"d84238c7-04cc-4c81-85bb-05055a338a44","added_by":"auto","created_at":"2025-09-19 16:36:21","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":38224,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/cfee38b48dd462d693e28c7d.png"},{"id":91732635,"identity":"2e6c08d1-1b50-48b9-bda1-2fa546674fd9","added_by":"auto","created_at":"2025-09-19 16:28:21","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":75622,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/0911770836f1e59716aef692.png"},{"id":91732863,"identity":"4d393097-87fd-47ec-8923-2f8128e8d737","added_by":"auto","created_at":"2025-09-19 16:36:21","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116931,"visible":true,"origin":"","legend":"","description":"","filename":"a1213b030d174d5bb5fd07b25e758ff21structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/581480dcbdfae767f3b01651.xml"},{"id":91732631,"identity":"c5f8bc69-3a92-4b84-97be-b75fc3aff5bf","added_by":"auto","created_at":"2025-09-19 16:28:21","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125115,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/85a41f07db767a8898b69f4c.html"},{"id":91732619,"identity":"b1a0cd4e-4e66-457f-9cb7-28a68b1cb290","added_by":"auto","created_at":"2025-09-19 16:28:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":120575,"visible":true,"origin":"","legend":"\u003cp\u003ePanel a shows the mean normalized pupil traces following 1 min (black), 2 min (red), 3 min (blue), or 4 min (green) of dark-adaption. Panel b plots mean (±SEM) normalized MPC (red triangles) and PIPR (blue circles) as a function of the dark-adaptation time.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/1ca1b04bc53080b7666d10cb.png"},{"id":91732620,"identity":"35934af4-84c2-4e4e-a8e0-62f5672571d7","added_by":"auto","created_at":"2025-09-19 16:28:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":178780,"visible":true,"origin":"","legend":"\u003cp\u003eBaseline pupil diameter (a), MPC (b), and PIPR (c) are plotted as a function of the subject’s age. Data are shown for the long wavelength (red circles) and short wavelength (blue circles) stimuli and are fit with linear regression lines.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/a7778504c68ec6e8c282295e.png"},{"id":91732621,"identity":"d37140be-f033-491f-a8f7-02a146a3b9d0","added_by":"auto","created_at":"2025-09-19 16:28:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":151325,"visible":true,"origin":"","legend":"\u003cp\u003eTest-retest repeatability for the baseline pupil size is shown in the form of Bland-Altman plot. Each circle represents the difference between the two tests as a function of the mean of the two tests. The solid line represents the mean difference between tests for the 27 subjects and the dashed lines present the 95% limits of repeatability.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/74e405d82df5a17b457acbc2.png"},{"id":91732861,"identity":"e0f71d19-af33-43a2-8697-3ffcdb60ba1d","added_by":"auto","created_at":"2025-09-19 16:36:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":136655,"visible":true,"origin":"","legend":"\u003cp\u003eTest-retest repeatability for the MPC elicited by the red (a) and blue (b) stimuli shown in the form of Bland-Altman plot. Panel C shows test-retest repeatability for the PIPR (blue). All other conventions are as in Fig. 3.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/1ee6a753c3a294cc558651e1.png"},{"id":96105943,"identity":"f2dea22d-7f50-46fc-b13a-c0058e73a461","added_by":"auto","created_at":"2025-11-17 16:12:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":868843,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7383219/v1/9042a8b5-2338-40e6-85d9-a83339bcf69e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A rapid pupillometry protocol for clinical use: Effect of age and test-retest repeatability","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePupil size is adaptively modulated by the iris sphincter and dilator muscles to control the amount of light entering the eye. Although several factors affect pupil size, light stimulation is the primary driver and the response of the pupil to changes in illumination has been termed the pupillary light reflex (PLR). The PLR is largely mediated by intrinsically photosensitive retinal ganglion cells (ipRGCs) that also receive inputs from rod and cone photoreceptors [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Given that the PLR is dependent on the function of outer- and inner-retinal neurons, as well as afferent and efferent post-retina pathways, the PLR has become of interest in Ophthalmology and Neurology. Pupillometric assessments have been reported in patients with acquired [\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8 CR9\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and inherited [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] ocular dysfunction. Beyond the retina, pupillometry has been applied to study central nervous system function in conditions including traumatic brain injury [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], Alzheimer\u0026rsquo;s disease [\u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and Parkinson\u0026rsquo;s disease [\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTraditionally, clinical assessment of the pupil has been performed qualitatively using a penlight (e.g., static measurement of pupil diameter under light-adapted conditions or with the \u0026ldquo;swinging flashlight test\u0026rdquo; to detect relative afferent pupillary defects). In recent decades, however, commercially available infrared videography systems have been developed that can provide quantitative measures of pupil size and reactivity. These infrared systems permit assessing pupil dynamics under both light- and dark-adapted conditions, which has become of interest in the clinical assessment of patients who have cone and rod photoreceptor dystrophies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The PLR elicited by a high-luminance, short-wavelength stimulus has been of particular interest, as this combination allows assessment of inner retinal function by stimulating ipRGCs. Specifically, the PLR elicited by a brief (\u0026le;\u0026thinsp;1 s), high luminance, short-wavelength stimulus is characterized by a rapid, transient constriction that is thought to be rod- and cone-mediated [\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The transient constriction is followed by a slow, sustained constriction that is often referred to as the post-illumination pupil response (PIPR) and is believed to be generated by melanopsin activation within ipRGCs [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite the promise of clinical pupillometry, the technique has largely been restricted to research applications in academic medical centers. Wider application has, in large part, been hampered by instrument cost, portability, and burdensome test lengths. There have been attempts to create portable pupillometers, including implementation using smart phones [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and other devices [\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], but none are used routinely in clinical practice. These portable pupillometry devices differ considerably in their ease of use, data analysis capabilities, and ability to elicit PLRs mediated by the rod-, cone-, and melanopsin-pathways.\u003c/p\u003e\u003cp\u003eRecently, a portable, hand-held instrument for retinal electrophysiology (RETeval, LKC Technologies Inc., Gaithersburg, MD, USA) has been adapted to measure the PLR [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The instrument can produce stimuli that are high luminance and nearly full field in size, which are important features for eliciting a robust PIPR. The first report with this device [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] elicited pupil responses using low (50 cd/m\u003csup\u003e2\u003c/sup\u003e) and high (316 cd/m\u003csup\u003e2\u003c/sup\u003e) luminance flashes of blue light (470 nm) and the pupil size was recorded using built-in software, allowing for quantitative analyses. This protocol used only 3 minutes of dark-adaptation (DA), which expedited testing. Data from this small clinical study indicated that the maximum transient constriction differed between healthy control subjects (N\u0026thinsp;=\u0026thinsp;5) and patients with optic neuropathy (N\u0026thinsp;=\u0026thinsp;10); the PIPR was not reported. This report was followed by a second study [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] that obtained pupil responses from 32 healthy, young subjects (20\u0026ndash;24 years of age) using 100 cd/m\u003csup\u003e2\u003c/sup\u003e red and blue flashes of light. The DA period was 10 minutes; baseline pupil size and maximum transient constriction were reported. Robust pupil responses were elicited with the RETeval and the responses were in excellent agreement with a lab-based pupillometer (Iriscorder Dual, Hamamatsu Photonics, Hamamatsu, Japan). Most recently, the RETeval was used to examine pupil responses in patients with early onset high myopia (N\u0026thinsp;=\u0026thinsp;14) and heathy emmetropic controls (N\u0026thinsp;=\u0026thinsp;12) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Following 10 min of DA, 250 cd/m\u003csup\u003e2\u003c/sup\u003e red and blue flashes were presented and the baseline, maximum transient constriction, and PIPR were recorded. Reduced PIPRs were observed in myopic subjects who had MYP-26 gene variants.\u003c/p\u003e\u003cp\u003eAll three studies show promise for the clinical application of portable pupillometry with the RETeval. However, the study protocols tended to be lengthy, data were obtained primarily from young subjects, and measures of test-retest repeatability were not performed. An additional limitation is that these studies used a constant flash luminance to elicit pupil constrictions, regardless of the subject\u0026rsquo;s baseline pupil size. This results in different amounts of light entering the eye, which may contribute to the inter-subject variability in these previous reports. Additionally, the studies were focused on the maximum transient constriction following the flash, with only one study reporting the PIPR. Thus, the purpose of the present study was to build on these previous reports to develop a rapid, clinically-applicable chromatic pupillometry protocol. In contrast to previous studies, stimuli were defined in Trolands, which accounted for differences in baseline pupil sizes among individuals, ensuring the same amount of light entered the eye of each subject. In Experiment I, the effect of DA duration was studied to define the minimum time needed to achieve maximum responses. In Experiment II, the effects of age on the transient and PIPR components of the PLR were determined. In Experiment III, test-retest repeatability of the transient and PIPR components was studied.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSubjects\u003c/h2\u003e\u003cp\u003eThirty-seven healthy subjects were recruited from the University of Illinois Chicago, Department of Ophthalmology and Visual Sciences. The subjects were 20 to 61 years old (average 42\u0026thinsp;\u0026plusmn;\u0026thinsp;13; 20 females). Subjects were excluded if there was a history of, or if they presented with, diabetes, glaucoma, optic neuropathy, or any other ophthalmic condition known to affect retinal or pupil function. Additionally, subjects with asymmetrically shaped pupils were excluded and no subject had a history of a neurologic event (cerebrovascular stroke or transient ischemic attack). The research followed the tenets of the Declaration of Helsinki and was approved by an institutional review board of the University of Illinois Chicago. All subjects provided written informed consent.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eApparatus, stimuli, and procedure\u003c/h3\u003e\n\u003cp\u003ePupillometry was performed on the right eye of each subject using the RETeval device. The stimuli consisted of red (621 nm) and blue (470 nm) LED-generated flashes. The flash duration was 500 milliseconds and a constant stimulus flash retinal illuminance (12,000 Td) was generated by monitoring the subject\u0026rsquo;s pupil size using a built-in IR-sensitive camera and adjusting luminance accordingly. The IR camera has 0.08 mm/pixel resolution at the iris and a 28.3 Hz recording rate. Pupil diameters were exported using an extraction tool supplied by the manufacturer. The waveforms were processed using a custom script programmed in MATLAB R2021b (MathWorks Inc., Natick, MA), which allowed for semi-automated analysis as described elsewhere [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. For ease of processing, data were extracted and analyzed, but the output of the pupil metrics recorded in this study can now be obtained directly from the device\u0026rsquo;s display or from automatically generated accompanying PDF reports. The automatically generated reports were not available at the time the study was initiated, which necessitated the use of the off-line semi-automated approach. Subsequent comparison of the semi-automated analysis with the manufacturer\u0026rsquo;s automatically processed values found excellent consistency.\u003c/p\u003e\u003cp\u003eMeasurement of the PLR elicited by the red flash always preceded the blue flash with an inter-stimulus interval (ISI) of at least 30 sec. Typically, a single PLR was obtained, but in cases of blinks or noise, the PLR measurement was repeated with an ISI of at least 60 sec. Baseline pupil size (BL) was defined as the median pupil size (mm) during the 1-sec preceding each stimulus onset, and each PLR sweep was normalized by the BL. Maximum pupil constriction (MPC) was defined as the difference between BL and the minimum pupil size after stimulus onset (normalized units). The post-illumination pupil response (PIPR) was defined as difference between the BL and the median pupil size at 6\u0026ndash;8 sec after stimulus offset (normalized units).\u003c/p\u003e\u003cp\u003eThe effect of DA duration was examined in Exp I in 13 subjects (44\u0026thinsp;\u0026plusmn;\u0026thinsp;13.1 years; 7 female). For this experiment, PLRs were only elicited with the blue stimulus. The subjects were dark-adapted for randomly assigned duration of 1, 2, 3, or 4 min, then exposed to the blue flash. After recording, the subject was exposed to room light for least 2 min before the next randomly selected DA period began. The effect of age on BL, MPC, and PIPR was evaluated in Exp II in 37 subjects. Exp III examined test-retest repeatability using Bland-Altman analysis. A subset of 28 subjects (41.8\u0026thinsp;\u0026plusmn;\u0026thinsp;11.3 years; 15 females) was tested twice using the same protocol developed in Exp II. The interval between two tests ranged from approximately 15 minutes to 292 days (mean test interval of 53.7\u0026thinsp;\u0026plusmn;\u0026thinsp;105.8 days).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea shows the mean normalized pupil traces following 1 min (black), 2 min (red), 3 min (blue), or 4 min (green) of DA. The PLRs were characterized by an initial transient constriction (\u0026ldquo;MPC;\u0026rdquo; rod- and cone-mediated) followed by a sustained constriction that persisted for several seconds after stimulus offset (\u0026ldquo;PIPR;\u0026rdquo; melanopsin-mediated). It is clear that both the MPC and PIPR increased from 1 to 3 min of DA, with relatively little change from 3 to 4 min. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb plots the mean (\u0026plusmn;\u0026thinsp;SEM) MPC (red) and PIPR (blue) amplitudes as a function of DA duration. A two-parameter exponential growth to maximum function provide a good description of the data (solid lines). The MPC increased in amplitude from 1 to 2 min of DA and was essentially constant thereafter. The PIPR increased in amplitude from 1 to 3 min of DA, and was essentially constant thereafter. Of note, there was a clear difference in MPC and PIPR amplitude following 1 min DA, a small difference following 2 min DA, and little difference following 3 or 4 min DA. Similar values of MPC and PIPR are indicative of minimal return to baseline at 6\u0026ndash;8 sec following the flash due to a robust melanopsin-mediated PIPR (apparent in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Based on these results, a 3-min DA period was used for Exp II and III.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e plots BL (a), MPC (b), and PIPR (c) as a function of the subject\u0026rsquo;s age; data are shown for red and blue stimuli and are fit with linear regression lines (dashed). BLs prior to the red and blue flashes were nearly identical for a given subject, with the red and blue regression lines essentially overlapping (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). There was a trend for BL to decrease with increasing age (red and blue MPC r \u0026gt;-0.23, p\u0026thinsp;\u0026gt;\u0026thinsp;0.16), but this was not statistically significant. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb shows that the MPC elicited by the blue stimulus was larger than that elicited by the red stimulus. There was a trend for the blue MPC to decrease with increasing age (r = -0.32, p\u0026thinsp;=\u0026thinsp;0.054), but this was not statistically significant; red MPC was independent of age (r\u0026thinsp;=\u0026thinsp;0.003, p\u0026thinsp;=\u0026thinsp;0.99). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec shows that the PIPR elicited by the red stimulus was negligible (mean red PIPR\u0026thinsp;=\u0026thinsp;0.06) and there was no significant correlation with age (r\u0026thinsp;=\u0026thinsp;0.26, p\u0026thinsp;=\u0026thinsp;0.12). The blue stimulus elicited a large PIPR (mean blue PIPR\u0026thinsp;=\u0026thinsp;0.46), but there was no significant correlation with age (r = -0.01, p\u0026thinsp;=\u0026thinsp;0.96).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePrevious work identified weak effects of sex on the PLR, with females having slightly larger MPC under dark-adapted conditions, compared to males [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. A possible effect of sex on BL, MPC, and PIPR was evaluated in the present data set. Two-way ANOVAs were conducted with stimulus wavelength and subject sex as main effects. ANOVA indicated no significant effect of sex on BL, MPC, or PIPR (F\u0026thinsp;\u0026lt;\u0026thinsp;0.64, p\u0026thinsp;\u0026gt;\u0026thinsp;0.43) and no interaction between sex and stimulus wavelength for any measure (F\u0026thinsp;\u0026lt;\u0026thinsp;3.34, p\u0026thinsp;\u0026gt;\u0026thinsp;0.07).\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e plots the difference in BL for the two repeat tests as a function of the mean of the two tests in the form of a Bland-Altman plot. Data are only shown for the BL preceding the red stimulus. The mean BL difference for the two tests was 0.04 mm (0.7%; solid blue line) and the upper and lower limits of repeatability (\u0026plusmn;\u0026thinsp;95%; dashed lines) were +\u0026thinsp;0.98 mm and \u0026minus;\u0026thinsp;0.90 mm, respectively. The difference between the two tests was not significantly correlated with the overall BL size (r\u0026thinsp;=\u0026thinsp;0.01, p\u0026thinsp;=\u0026thinsp;0.96).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb represent repeat measurements of the red and blue MPC in the same Bland-Altman format as Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea shows the mean difference between tests for the red MPC was 0.01, with an upper limit of repeatability of 0.10 and a lower limit of -0.08. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb shows the mean difference between tests for the blue MPC was 0.01, with an upper limit of repeatability of 0.10 and a lower limit of -0.09. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec shows the mean difference between the PIPR measurements for the blue stimulus. The mean difference between tests was \u0026minus;\u0026thinsp;0.001, with upper and lower limits of repeatability of +\u0026thinsp;0.11 and \u0026minus;\u0026thinsp;0.11, respectively. The difference between the two tests was not significantly correlated with the overall MPC or PIPR size (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.37)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe purpose of this study was to develop and evaluate a rapid, clinically-applicable pupillometry protocol using a commercially available, portable, handheld instrument. The primary findings were 1) a 3-min DA period is sufficient to elicit robust MPCs and PIPRs; 2) there is no statistically significant relationship between age and BL, MPC, or PIPR in our sample of subjects across the ages of 26\u0026ndash;61 years; 3) test-retest repeatability was good, with differences within approximately 10% observed. The total test time, including DA, for one eye was approximately 5 min.\u003c/p\u003e\u003cp\u003eDA periods of 10 to 15 min are commonly employed in pupillometry studies [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Although less demanding than the ISCEV standard 20 min DA for full-field electroretinography [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], DA remains a burden in clinical testing. Data of the present study show that 3 min is sufficient to elicit large MPCs and PIPRs in healthy, visually-normal individuals. This finding is consistent with the conclusion of Bindiganavale [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] who reported maximum pupillary constrictions following three minutes of DA. We observed minimal differences in pupil response between 3 and 4 min, and therefore used 3 min DA to expedite testing. Although the stimulus parameters selected for this study elicited large MPCs and PIPRs after only 3 min of DA, a 3-min DA period may not elicit maximum MPCs and PIPRs for other stimuli. For example, Wang et al. [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] reported that the PIPR increased over a time-course of approximately 20 min of DA for a 100 cd/m\u003csup\u003e2\u003c/sup\u003e flash (1 sec; 465 nm). It is also important to note that that present findings were obtained only from healthy, visually-normal subjects and that DA dynamics may differ in patient populations.\u003c/p\u003e\u003cp\u003eSomewhat surprisingly, there was relatively little effect of age on the pupil metrics that were assessed in the present study. BL decreased by 0.17 mm with each increasing decade of age, but this relationship did not achieve statistical significance. \u0026ldquo;Senile miosis\u0026rdquo; is a well-known effect of aging [\u003cspan additionalcitationids=\"CR50 CR51\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. It is possible that the effect of age on BL may have been more apparent in the present dataset if a larger number of subjects older than 60 years of age were included. Studies reporting the effect of age on the human PIPR have been inconsistent [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Age-related loss of ipRGC subtypes has been reported in the human retina [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], but the loss appears most prominently after the age of 70. Rather than loss of ipRGCs, reduced blue light sensitivity due to cataract formation may be a more influential factor to consider when interpreting chromatic pupillometry results across age.\u003c/p\u003e\u003cp\u003eTest-retest repeatability in the present sample of subjects was good, with differences less than approximately 10% observed, defined by Bland-Altman analyses. Herbst et al. [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] reported intraclass coefficients (ICCs) for maximum constrictions elicited by red and blue flashes measured with a custom pupilometer to be 0.7 to 0.8, which is similar to our MPC ICC value of 0.71. Li et al. [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] reported ICCs of 0.84 to 0.94 for the PIPR elicited by full-field blue flashes, which is similar to our PIPR ICC of 0.97. Of note, the present study is underpowered to assess test-retest repeatability within a narrow confidence interval. To define repeatability within a 10% confidence interval, for example, 190 subjects would be needed. Our sample size of 27 subjects provides a much larger confidence interval of 27%. Nevertheless, the data provided herein represents a first step toward a potential standardized approach to rapid portable pupillometry.\u003c/p\u003e\u003cp\u003eIn summary, we provide evidence that a short-duration, clinically applicable, chromatic pupillometry test can be used to assess function in healthy individuals. The test is administered quickly (approximately 5 min for one eye), is well tolerated, and repeatable.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by JCP. The first draft of the manuscript was written by JJM and JCP. Both authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest. The research was supported by the National Institutes of Health research grants R01EY026004 (JM), P30EY001792 (core grant), an unrestricted departmental grant from Research to Prevent Blindness.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe studies were performed in accordance with the tenants of the Declaration of Helsinki, institutional review board approval was obtained at the University of Illinois at Chicago, and the experiments were undertaken with the understanding and written consent of each subject.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement on the welfare of animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo animal experiments were performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe subjects consented to participate in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrant Information:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNational Institutes of Health research grants R01EY026004 (JM), P30EY001792 (core grant), an unrestricted departmental grant from Research to Prevent Blindness.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSpitschan M (2019) Photoreceptor inputs to pupil control. J Vis 19(9):5\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuler AD, Ecker JL, Lall GS, Haq S, Altimus CM, Liao HW, Barnard AR, Cahill H, Badea TC, Zhao H, Hankins MW, Berson DM, Lucas RJ, Yau KW, Hattar S (2008) Melanopsin cells are the principal conduits for rod-cone input to non-image-forming vision. Nature 453(7191):102\u0026ndash;105\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHall CA, Chilcott RP (2018) Eyeing up the Future of the Pupillary Light Reflex in Neurodiagnostics. Diagnostics 8 (1)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeigl B, Zele AJ, Fader SM, Howes AN, Hughes CE, Jones KA, Jones R (2012) The post-illumination pupil response of melanopsin-expressing intrinsically photosensitive retinal ganglion cells in diabetes. Acta Ophthalmol 90(3):e230\u0026ndash;234\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFerrari GL, Marques JL, Gandhi RA, Heller SR, Schneider FK, Tesfaye S, Gamba HR (2010) Using dynamic pupillometry as a simple screening tool to detect autonomic neuropathy in patients with diabetes: a pilot study. Biomed Eng Online 9:26\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaravanaki K, Davies AG, Hunt LP, Morgan MH, Baum JD (1994) Pupil size in diabetes. Arch Dis Child 71(6):511\u0026ndash;515\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark JC, Chen YF, Blair NP, Chau FY, Lim JI, Leiderman YI, Shahidi M, McAnany JJ (2017) Pupillary responses in non-proliferative diabetic retinopathy. Sci Rep 7:44987\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark JC, Moss HE, McAnany JJ (2016) The Pupillary Light Reflex in Idiopathic Intracranial Hypertension. Invest Ophthalmol Vis Sci 57(1):23\u0026ndash;29\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNajjar RP, Sharma S, Atalay E, Rukmini AV, Sun C, Lock JZ, Baskaran M, Perera SA, Husain R, Lamoureux E, Gooley JJ, Aung T, Milea D (2018) Pupillary Responses to Full-Field Chromatic Stimuli Are Reduced in Patients with Early-Stage Primary Open-Angle Glaucoma. Ophthalmology 125(9):1362\u0026ndash;1371\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKelbsch C, Maeda F, Strasser T, Blumenstock G, Wilhelm B, Wilhelm H, Peters T (2016) Pupillary responses driven by ipRGCs and classical photoreceptors are impaired in glaucoma. Graefes Arch Clin Exp Ophthalmol 254(7):1361\u0026ndash;1370\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKawasaki A, Crippa SV, Kardon R, Leon L, Hamel C (2012) Characterization of pupil responses to blue and red light stimuli in autosomal dominant retinitis pigmentosa due to NR2E3 mutation. Invest Ophthalmol Vis Sci 53(9):5562\u0026ndash;5569\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCollison FT, Park JC, Fishman GA, McAnany JJ, Stone EM (2015) Full-Field Pupillary Light Responses, Luminance Thresholds, and Light Discomfort Thresholds in CEP290 Leber Congenital Amaurosis Patients. Invest Ophthalmol Vis Sci 56(12):7130\u0026ndash;7136\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCollison FT, Park JC, Fishman GA, Stone EM, McAnany JJ (2016) Two-color pupillometry in enhanced S-cone syndrome caused by NR2E3 mutations. Doc Ophthalmol 132(3):157\u0026ndash;166\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKawasaki A, Collomb S, Leon L, Munch M (2014) Pupil responses derived from outer and inner retinal photoreception are normal in patients with hereditary optic neuropathy. Exp Eye Res 120:161\u0026ndash;166\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHyde RA, Kratunova E, Park JC, McAnany JJ (2022) Cone pathway dysfunction in Jalili syndrome due to a novel familial variant of CNNM4 revealed by pupillometry and electrophysiologic investigations. Ophthalmic Genet 43(2):268\u0026ndash;276\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarboni MTS, Szell N, Sohajda Z, Feher T (2024) Pupillary Light Reflex Reveals Melanopsin System Alteration in the Background of Myopia-26, the Female Limited Form of Early-Onset High Myopia. Invest Ophthalmol Vis Sci 65(8):6\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThe Brain Trauma Foundation. The American Association of Neurological Surgeons (2000) The Joint Section on Neurotrauma and Critical Care. Pupillary diameter and light reflex. J Neurotrauma 17(6\u0026ndash;7):583\u0026ndash;590\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYuhas PT, Shorter PD, McDaniel CE, Earley MJ, Hartwick AT (2017) Blue and Red Light-Evoked Pupil Responses in Photophobic Subjects with TBI. Optometry Vis science: official publication Am Acad Optometry 94(1):108\u0026ndash;117\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMostafa J, Porter J, Queener HM, Ostrin LA (2021) Intrinsically photosensitive retinal ganglion cell-driven pupil responses in patients with traumatic brain injury. Vis Res 188:174\u0026ndash;183\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFrost S, Kanagasingam Y, Sohrabi H, Bourgeat P, Villemagne V, Rowe CC, Macaulay SL, Szoeke C, Ellis KA, Ames D, Masters CL, Rainey-Smith S, Martins RN, Group AR (2013) Pupil response biomarkers for early detection and monitoring of Alzheimer's disease. Curr Alzheimer Res 10(9):931\u0026ndash;939\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTales A, Troscianko T, Lush D, Haworth J, Wilcock GK, Butler SR (2001) The pupillary light reflex in aging and Alzheimer's disease. Aging 13(6):473\u0026ndash;478\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOh AJ, Amore G, Sultan W, Asanad S, Park JC, Romagnoli M, La Morgia C, Karanjia R, Harrington MG, Sadun AA (2019) Pupillometry evaluation of melanopsin retinal ganglion cell function and sleep-wake activity in pre-symptomatic Alzheimer's disease. PLoS ONE 14(12):e0226197\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRomagnoli M, Stanzani Maserati M, De Matteis M, Capellari S, Carbonelli M, Amore G, Cantalupo G, Zenesini C, Liguori R, Sadun AA, Carelli V, Park JC, La Morgia C (2020) Chromatic Pupillometry Findings in Alzheimer's Disease. Front NeuroSci 14:780\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLa Morgia C, Mitolo M, Romagnoli M, Stanzani Maserati M, Evangelisti S, De Matteis M, Capellari S, Bianchini C, Testa C, Vandewalle G, Santoro A, Carbonelli M, D'Agati P, Filardi M, Avanzini P, Barboni P, Zenesini C, Baccari F, Liguori R, Tonon C, Lodi R, Carelli V (2023) Multimodal investigation of melanopsin retinal ganglion cells in Alzheimer's disease. Ann Clin Transl Neurol 10(6):918\u0026ndash;932\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGiza E, Fotiou D, Bostantjopoulou S, Katsarou Z, Karlovasitou A (2011) Pupil light reflex in Parkinson's disease: evaluation with pupillometry. Int J Neurosci 121(1):37\u0026ndash;43\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoyce DS, Feigl B, Kerr G, Roeder L, Zele AJ (2018) Melanopsin-mediated pupil function is impaired in Parkinson's disease. Sci Rep 8(1):7796\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeigl B, Dumpala S, Kerr GK, Zele AJ (2020) Melanopsin Cell Dysfunction is Involved in Sleep Disruption in Parkinson's Disease. J Parkinson's disease 10(4):1467\u0026ndash;1476\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTabashum T, Zaffer A, Yousefzai R, Colletta K, Jost MB, Park Y, Chawla J, Gaynes B, Albert MV, Xiao T (2021) Detection of Parkinson's Disease Through Automated Pupil Tracking of the Post-illumination Pupillary Response. Front Med 8:645293\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGaynes BI, Zaffer A, Yousefzai R, Chazaro-Cortes M, Colletta K, Kletzel SL, Jost MB, Park Y, Chawla J, Albert MV, Xiao T (2022) Variable abnormality of the melanopsin-derived portion of the pupillary light reflex (PLR) in patients with Parkinson's disease (PD) and parkinsonism features. Neurol sciences: official J Italian Neurol Soc Italian Soc Clin Neurophysiol 43(1):349\u0026ndash;356\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCharng J, Jacobson SG, Heon E, Roman AJ, McGuigan DB 3rd, Sheplock R, Kosyk MS, Swider M, Cideciyan AV (2017) Pupillary Light Reflexes in Severe Photoreceptor Blindness Isolate the Melanopic Component of Intrinsically Photosensitive Retinal Ganglion Cells. Invest Ophthalmol Vis Sci 58(7):3215\u0026ndash;3224\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcAnany JJ, Park JC, Fishman GA, Collison FT (2020) Full-Field Electroretinography, Pupillometry, and Luminance Thresholds in X-Linked Retinoschisis. Invest Ophthalmol Vis Sci 61(6):53\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark JC, Moura AL, Raza AS, Rhee DW, Kardon RH, Hood DC (2011) Toward a clinical protocol for assessing rod, cone, and melanopsin contributions to the human pupil response. Invest Ophthalmol Vis Sci 52(9):6624\u0026ndash;6635\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKostic C, Crippa SV, Martin C, Kardon RH, Biel M, Arsenijevic Y, Kawasaki A (2016) Determination of Rod and Cone Influence to the Early and Late Dynamic of the Pupillary Light Response. Invest Ophthalmol Vis Sci 57(6):2501\u0026ndash;2508\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdhikari P, Zele AJ, Feigl B (2015) The Post-Illumination Pupil Response (PIPR). Invest Ophthalmol Vis Sci 56(6):3838\u0026ndash;3849\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePhilibert M, Milea D (2024) Basics, benefits, and pitfalls of pupillometers assessing visual function. Eye (Lond) 38(12):2415\u0026ndash;2421\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKardon R, Anderson SC, Damarjian TG, Grace EM, Stone E, Kawasaki A (2011) Chromatic pupillometry in patients with retinitis pigmentosa. Ophthalmology 118(2):376\u0026ndash;381\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcAnany JJ, Smith BM, Garland A, Kagen SL (2018) iPhone-based Pupillometry: A Novel Approach for Assessing the Pupillary Light Reflex. Optometry Vis science: official publication Am Acad Optometry 95(10):953\u0026ndash;958\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTan HH, Lee KC, Chen YR, Huang YC, Ke RS, Horng GJ, Chen KT (2025) Using smartphone pupillometer application to measure pupil size and light reflex: An unsuccessful prototype and analysis of the causes of failure. Medicine 104(9):e41682\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEmelifeonwu JA, Reid K, Rhodes JK, Myles L (2018) Saved by the Pupillometer! - A role for pupillometry in the acute assessment of patients with traumatic brain injuries? Brain Injury 32(5):675\u0026ndash;677\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJehu DA, Bolgla LA, Armas S, Dutton F (2025) Assessing the Inter-Rater and Inter-Trial Reliability of the NeurOptics Pupillary Light Response-3000 Pupillometer. Int J sports Phys therapy 20(2):157\u0026ndash;167\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNajjar RP, Rukmini AV, Finkelstein MT, Nusinovici S, Mani B, Nongpiur ME, Perera S, Husain R, Aung T, Milea D (2023) Handheld chromatic pupillometry can accurately and rapidly reveal functional loss in glaucoma. Br J Ophthalmol 107(5):663\u0026ndash;670\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKohnen T, Terzi E, Buhren J, Kohnen EM (2003) Comparison of a digital and a handheld infrared pupillometer for determining scotopic pupil diameter. J Cataract Refract Surg 29(1):112\u0026ndash;117\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBindiganavale MP, Moss HE (2021) Development and Implementation of a Handheld Pupillometer for Detection of Optic Neuropathies. Curr Eye Res 46(9):1432\u0026ndash;1435\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAsakawa K, Imai M, Ohta M, Kawata N, Kawatsu N, Ishikawa H (2023) Pupil assessment with a new handheld pupillometer in healthy subjects. Int Ophthalmol 43(1):51\u0026ndash;61\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIshikawa M (2021) Clinical factors affecting pupillary light reflex parameters: a single-centre, cross-sectional study. Ophthalmic Physiol Opt 41(5):952\u0026ndash;960\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFan X, Hearne L, Lei B, Miles JH, Takahashi N, Yao G (2009) Weak gender effects on transient pupillary light reflex. Auton neuroscience: basic Clin 147(1\u0026ndash;2):9\u0026ndash;13\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang B, Shen C, Zhang L, Qi L, Yao L, Chen J, Yang G, Chen T, Zhang Z (2015) Dark adaptation-induced changes in rod, cone and intrinsically photosensitive retinal ganglion cell (ipRGC) sensitivity differentially affect the pupil light response (PLR). Graefes Arch Clin Exp Ophthalmol 253(11):1997\u0026ndash;2005\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRobson AG, Frishman LJ, Grigg J, Hamilton R, Jeffrey BG, Kondo M, Li S, McCulloch DL (2022) ISCEV Standard for full-field clinical electroretinography (2022 update). Doc Ophthalmol Adv Ophthalmol 144(3):165\u0026ndash;177\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKornzweig A (1954) Physiological effects of age on the visual process. Sight Sav Rev 24:130\u0026ndash;138\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBirren JE, Casperson RC, Botwinick J (1950) Age Changes in Pupil Size. J Gerontol 5(3):216\u0026ndash;221\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIE L (1979) Pupillary changes related to age. Topic in Neuro-Ophthalmology\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRobertson GW, Yudkin J (1944) Effect of age upon dark adaptation. J Physiol 103(1):1\u0026ndash;8\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen Y, Pinto AA, Paulsen AJ, Schubert CR, Hancock LM, Klein BE, Klein R, Cruickshanks KJ (2019) The Post-illumination Pupil Response (PIPR) Is Associated With Cognitive Function in an Epidemiologic Cohort Study. Front Neurol Volume 10\u0026ndash;2019\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHerbst K, Sander B, Lund-Andersen H, Broendsted AE, Kessel L, Hansen MS, Kawasaki A (2012) Intrinsically photosensitive retinal ganglion cell function in relation to age: a pupillometric study in humans with special reference to the age-related optic properties of the lens. BMC Ophthalmol 12:4\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKlevens AM, Taylor ML, Wescott DL, Gamlin PD, Franzen PL, Hasler BP, Siegle G, Roecklein KA (2024) The role of retinal irradiance estimates in melanopsin-driven retinal responsivity: a reanalysis of the post-illumination pupil response in seasonal affective disorder. Sleep 47 (9)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEsquiva G, Lax P, Perez-Santonja JJ, Garcia-Fernandez JM, Cuenca N (2017) Loss of Melanopsin-Expressing Ganglion Cell Subtypes and Dendritic Degeneration in the Aging Human Retina. Front Aging Neurosci 9:79\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHerbst K, Sander B, Milea D, Lund-Andersen H, Kawasaki A (2011) Test-retest repeatability of the pupil light response to blue and red light stimuli in normal human eyes using a novel pupillometer. Front Neurol 2:10\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLei S, Goltz HC, Chandrakumar M, Wong AM (2015) Test-retest reliability of hemifield, central-field, and full-field chromatic pupillometry for assessing the function of melanopsin-containing retinal ganglion cells. Invest Ophthalmol Vis Sci 56(2):1267\u0026ndash;1273\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"documenta-ophthalmologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"doop","sideBox":"Learn more about [Documenta Ophthalmologica](http://link.springer.com/journal/10633)","snPcode":"10633","submissionUrl":"https://submission.nature.com/new-submission/10633/3","title":"Documenta Ophthalmologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7383219/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7383219/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose: \u003c/strong\u003ePupillometry is most commonly performed in laboratory settings using specialized, non-portable instruments that require lengthy test protocols. The purpose of this study was to develop and evaluate a rapid, clinically-applicable pupillometry protocol using a commercially available, portable, handheld instrument.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Thirty-seven healthy individuals (ages 21- 61 years) participated in three experiments. In each experiment, the pupillary light reflex (PLR) was elicited by full-field, 500-ms chromatic flashes (470 nm and 621 nm; 12,000 Td). Experiment I evaluated the minimum dark adaptation (DA) time needed to achieve maximum PLRs. Experiment II determined the effect of age. Experiment III estimated PLR test-retest repeatability. For all experiments, baseline pupil size (BL; 1 sec before flash onset), maximum pupil constriction (MPC) following the flash, and post-illumination pupillary response (PIPR; median size 6 - 8 sec after flash offset) were quantified.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eExperiment I showed that from 1 - 3 min of DA, BL and MPC increased slightly (0.27 mm and 5%, respectively), whereas the PIPR increased considerably (17%). The responses did not change appreciably after 3 min, therefore a 3-min DA period was used for Experiments II and III. Experiment II showed a trend for BL and MPC to decrease with age, but correlations with age were not statistically significant (all p \u0026gt; 0.05). PIPR was independent of age (r = -0.01; p = 0.96). Experiment III showed test-retest repeatability of approximately 1 mm for BL, and 10% for MPC and PIPR, indicating good repeatability.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e The proposed approach is useful for measuring the MPC and PIPR across a broad range of ages and baseline pupil sizes. Given the device portability and short test duration (approximately 5 minutes including DA), this approach has promising clinical utility.\u003c/p\u003e","manuscriptTitle":"A rapid pupillometry protocol for clinical use: Effect of age and test-retest repeatability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-19 16:28:16","doi":"10.21203/rs.3.rs-7383219/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-09T14:11:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T10:16:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8067675461857628233727448246499481837","date":"2025-10-08T06:26:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T19:37:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177491906446806751071136522596393826804","date":"2025-09-16T05:57:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-12T16:42:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-20T11:46:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-20T11:46:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Documenta Ophthalmologica","date":"2025-08-15T17:25:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"documenta-ophthalmologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"doop","sideBox":"Learn more about [Documenta Ophthalmologica](http://link.springer.com/journal/10633)","snPcode":"10633","submissionUrl":"https://submission.nature.com/new-submission/10633/3","title":"Documenta Ophthalmologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ed0b4aab-6db1-47e6-a111-3aa5c138d40c","owner":[],"postedDate":"September 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:11:08+00:00","versionOfRecord":{"articleIdentity":"rs-7383219","link":"https://doi.org/10.1007/s10633-025-10068-5","journal":{"identity":"documenta-ophthalmologica","isVorOnly":false,"title":"Documenta Ophthalmologica"},"publishedOn":"2025-11-15 15:58:48","publishedOnDateReadable":"November 15th, 2025"},"versionCreatedAt":"2025-09-19 16:28:16","video":"","vorDoi":"10.1007/s10633-025-10068-5","vorDoiUrl":"https://doi.org/10.1007/s10633-025-10068-5","workflowStages":[]},"version":"v1","identity":"rs-7383219","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7383219","identity":"rs-7383219","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}