Cluster analysis of heart rate variability reveals subgroups with preserved and early- impaired autonomic regulation in amyotrophic lateral sclerosis

Cluster analysis of heart rate variability reveals subgroups with preserved and early- impaired autonomic regulation in amyotrophic lateral sclerosis | 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 Cluster analysis of heart rate variability reveals subgroups with preserved and early- impaired autonomic regulation in amyotrophic lateral sclerosis Juri Sawada, Yuki Nakayama, Keiichi Shimatani, Chiharu Matsuda, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7862037/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 May, 2026 Read the published version in BMC Neurology → Version 1 posted 10 You are reading this latest preprint version Abstract Background Patients with amyotrophic lateral sclerosis (ALS) occasionally exhibit autonomic nervous system dysregulation. We examined whether autonomic regulation differed across patients with ALS with varying severity and progression. Methods A total of 45 patients with ALS were enrolled and classified into three subgroups using cluster analysis. Heart rate variability was assessed using the maximum entropy method. The low-frequency (LF) and high-frequency (HF) components, LF/HF ratio (LF/HF), and heart rate (HR) were measured. Temporal changes in each parameter during rest, mental tasks, and post-task rest were evaluated. The values for all patients and subgroups were compared with those of 11 healthy control subjects. Between-group differences were evaluated at rest and using the Task/Rest and After/Task ratios, and within-group changes across the three phases were also analyzed, with non-parametric statistical tests applied and significance set at p < 0.05. Results Cluster analysis classified the patients into three groups: “Group 1: early-preserved group”, “Group 2: late-preserved group”, and “Group 3: late-impaired group”. Overall, the patients showed lower HF and higher LF/HF at rest than the controls, indicating parasympathetic hypoactivity and sympathetic predominance. Abnormalities were more prominent in Groups 1 and 3 than in Group 2. The former two groups showed blunted HF, LF/HF and HR responses during the tasks. The late-preserved group showed no difference in the Task/Rest ratios of HF, LF/HF and HR compared with the controls. Conclusion Autonomic regulatory functions differ depending on the severity and progression of ALS. Autonomic dysregulation in ALS may be associated with a decline in motor function. The presence of HRV abnormalities in early-preserved patients suggests that autonomic dysfunction may precede overt motor decline in rapidly progressing cases. This supports the hypothesis that autonomic dysregulation is not merely a late complication but a fundamental component of ALS pathophysiology. Recognizing HRV abnormalities from early stages may help identify patients at risk of faster progression and guide timely interventions. Future longitudinal studies are needed to confirm whether disease-modifying therapies can alter HRV trajectories and improve prognosis. amyotrophic lateral sclerosis heart rate variability autonomic function sympathetic hyperactivity sympathovagal imbalance Figures Figure 1 Figure 2 Figure 3 Introduction Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons. However, recent studies have indicated that it may be involved in multisystem degeneration affecting autonomic and sensory neurons [ 1 , 2 ]. Non-motor symptoms unrelated to motor neuron impairment are observed in 5%–80% of patients with ALS [ 3 ] and significantly reduce their quality of life [ 4 ]. Autonomic symptoms of ALS include cardiovascular, gastrointestinal, voiding, and sudomotor dysfunction [ 5 ]. Urinary and gastrointestinal symptoms are noted in 30% of patients with ALS [ 6 ]. While these autonomic dysfunctions may appear mild, some patients with tracheostomy and invasive ventilation (TIV) in the advanced stages may present prominent fluctuations of blood pressure and heart rate, known as “autonomic storm,” which can lead to sudden death [ 7 ]. This condition is caused by central sympathetic hyperactivity and downregulation of peripheral sympathetic receptor function, which may result in circulatory collapse [ 8 , 9 ]. Moreover, since unstable blood pressure is associated with disease progression [ 10 ], autonomic dysfunction is a significant concern in ALS. Recent studies have reported an imbalance between the sympathetic and parasympathetic systems in ALS [ 5 ]. Assessment of heart rate variability (HRV), a non-invasive measure of autonomic function, revealed that patients with ALS exhibited reduced autonomic activity with sympathetic predominance compared to healthy individuals [ 11 – 14 ]. Neurophysiological studies indicated that muscle sympathetic nerve activity (MSNA) and skin sympathetic nerve activity (SSNA) initially increased and then decreased with age and disease duration [ 15 , 16 ]. Secondary factors such as psychological stress, long-term ventilatory support, severe muscle atrophy, long-term bedridden state, and repetitive infections may contribute to sympathetic hyperactivity [ 10 , 17 ]. Although these autonomic abnormalities are evident, including sympathetic hyperactivity and concomitant/subsequent blunted vascular responses, the mechanisms and factors underlying these autonomic dysfunctions remain unclear. Focusing on patients with ALS at the stages before ventilator use, previous studies reported that decreased HRV was associated with decreased lung capacity and increased disease duration [ 18 , 19 ]. In contrast, a study found respiratory dysfunction was not associated with decreased parasympathetic activity [ 20 ]. Considering the heterogeneous progression of ALS, several additional measures that capture ALS progression must be considered. Although most studies examined autonomic function at rest, evaluating autonomic function in response to tasks may provide a more comprehensive assessment. Understanding the factors associated with autonomic dysregulation in ALS could lead to early and effective interventions before TIV implementation. This study examined whether autonomic dysregulation differs across patients with ALS with varying severity and progression using non-invasive HRV measurements. This study aimed to identify factors associated with autonomic dysregulation in ALS by assessing sympathetic and parasympathetic nervous system functions under task conditions. Methods Participants We enrolled 45 patients who visited the Tokyo Metropolitan Neurological Hospital between March 2017 and September 2025 and were diagnosed with sporadic ALS. Although the enrollment of patients was not consecutive, we enrolled patients who provided written informed consent to participate in the study. All patients were diagnosed with ALS according to the revised El Escorial criteria as “clinically definite,” “clinically probable,” “clinically probable-laboratory supported,” or “clinically possible” ALS [ 21 ]. Patients with a history of alcohol or tobacco abuse, arrhythmias, or peripheral neuropathy were excluded. There were no patients who were taking medications that could affect autonomic functions at the time of investigation. All the participants maintained sufficient cognitive function to meet the task requirements. The following clinical characteristics were assessed: sex, age at onset, age at evaluation, height, premorbid weight, body weight at diagnosis, body weight at evaluation, disease duration (months), onset region (bulbar, upper or lower limb), use of enteral nutrition (EN), use of non-invasive ventilation (NIV), the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) at evaluation [ 22 ], and seated forced vital capacity (FVC) at evaluation. Thereafter, body mass index (BMI) was calculated as weight (kg)/height (m) 2 . FVC was expressed as a percentage and calculated as follows: (measured FVC/predicted FVC) × 100. ΔALSFRS-R was calculated as follows: (48 ALSFRS-R score at evaluation)/disease duration (months). ΔFVC was calculated as (100 – measured FVC)/disease duration (months) and ΔBMI as (premorbid BMI – BMI at evaluation)/disease duration (months). Eleven healthy subjects (median 59.0 (IQR 10.5) years, eight males) served as the control population [ 23 ]. This study was approved by the ethics committee of the Tokyo Metropolitan Neurological Hospital (No. R03-019) and the Tokyo Metropolitan Institute of Medical Science (No. 22 − 19). This study was performed according to the ethical standards described in the latest version of the Declaration of Helsinki and the Ethical Guidelines for Clinical Research of the Tokyo Metropolitan Neurological Hospital. All participants provided written informed consent before participating in the study. Heart Rate Variability Measurement HRV measurements were conducted in the outpatient examination room between 13:00 and 17:00 after routine consultations. Patients lay on a bed, and a quiet environment was set up for HRV measurements in a relaxed state. The three-behavioral-state paradigm consisting of the ‘Rest,’ ‘Task,’ and ‘After’ phases was applied based on previous studies [ 23 – 25 ]. During the Rest phase, patients relaxed on a bed for 120 s. For the Task phase, a 60-second silent Serial Sevens Test was performed in patients’ minds at their own pace. Finally, during the After phase, patients were instructed to cease the calculation and relax again for 120 s. The Serial Sevens Test, a mental arithmetic task with a high cognitive load [ 26 ], was used to assess mental stress in patients [ 27 ]. In the present study, each patient performed calculations in mind from ‘100 minus 7,’ ‘93 minus 7,’ and so on. We verified task completion by asking the patient the final number after the measurement was completed. Control subjects performed a random number generation task during the Task phase, as previously reported [ 24 , 25 ]. Data on control subjects were obtained from previous independent studies conducted by one of the co-authors [ 23 ]. Although the tasks’ characteristics differed between patients and control subjects in this study, we judged that the two tasks had equivalent loads to mental tasks. Spectral analysis of the HRV, which is widely used to assess autonomic function, was conducted using an ECG monitor (RF-ECG2; GM3, Tokyo, Japan) attached to the chest. ECG data were recorded on a computer, and R-R intervals were analyzed using the maximum entropy method (MemCalc, GMS, Tokyo, Japan) [ 28 ]. This method allows the analysis of short, 30-second data segments [ 29 ] and was applied to the three-behavioral-state paradigm in this study [ 24 , 25 ]. MemCalc calculated the low-frequency (LF) and high-frequency (HF) components every two seconds by integrating power within frequency ranges of 0.04–0.15 Hz for LF and 0.15–0.4 Hz for HF. The heart rate (HR, bpm) was calculated from the R-R intervals [ 28 ]. Statistical Analysis We utilized Uniform Manifold Approximation and Projection (UMAP) and Ordering Points To Identify the Clustering Structure (OPTICS) algorithms to characterize the clinical state of patients with ALS [ 30 ]. Using UMAP, nine variables that reflect the severity and progression of ALS (age, disease duration, ALSFRS-R, FVC, BMI, ΔBMI, and three binary variables: bulbar onset, EN use, and NIV use) were reduced to a two-dimensional space, allowing for visualization of the data structure [ 29 ]. In the dataset used, the FVC values were missing for two people (5.7% of the total); therefore, we used the k-nearest neighbor (KNN) method to fill in the missing values [ 31 , 32 ]. A composite distance metric combining the Euclidean distance for continuous variables and the Hamming distance for binary variables was applied, with the five nearest neighbors specified for each data point and a minimum distance parameter of 0.01. OPTICS analyzes the density structures in the reduced data, identifies high-density regions based on a 5% minimum point threshold, and generates a reachability plot. Clustering structures were visualized using this plot, and distinct clusters were identified using the Density-Based Spatial Clustering of Applications with Noise (DBSCAN) [ 33 ]. The DBSCAN distance threshold was determined from the “elbow” of the KNN distance plot. After assigning the patients to clusters, nine variables were summarized for each cluster. Differences in distributions between clusters were assessed to characterize the disease states. The Shapiro-Wilk test was used to evaluate variable distributions, guiding the choice of statistical methods. The Kruskal-Wallis test was used to compare continuous data between clusters, whereas the chi-squared test was used to analyze categorical data. We compared the resting HRV and resting HR values of the following groups to evaluate the differences in autonomic nervous dysfunction between patients with ALS and control subjects: control subjects vs. all patients and control subjects vs. each cluster. The Mann–Whitney U test was used for comparison. Next, we compared the changes in HRV and HR values in the three phases (Rest, Task, and After) between control subjects, all patients with ALS, and each cluster. We used Friedman’s repeated-measures test for this analysis and Nemenyi’s test for post-hoc comparisons. Finally, we compared the changes in HRV and HR values using the ratio of values in the Task phase to the Rest phase (Task/Rest) and the After phase to the Task phase (After/Task). Comparisons between control subjects and all patients were performed using the Mann-Whitney U test. In contrast, comparisons between control subjects and each cluster were performed using the Kruskal–Wallis test and a post-hoc Dunn’s test. All statistical analyses were conducted using R software (version 4.4.0) with the Uwot and DBSCAN packages [ 34 ], with the significance level set at p < 0.05. Results Patient characteristics The clinical characteristics of the patients are summarized in Table 1. The median age at examination was 64.0 (IQR 15.0) years. While eleven patients were using NIV, all used it either only at night or intermittently during the day, and none were using it at the time of the examination. Among the 19 patients using EN, 18 used gastrostomy tubes, and one used a nasogastric tube; all patients were in stable condition without pain or wound inflammation following gastrostomy tube placement. We identified three subgroups (clusters) among the patients using the OPTICS clustering algorithm. The distribution of clusters is shown in Fig. 1 (a). In Fig. 1 (b), the clusters detected by OPTICS are visualized by performing UMAP on a two-dimensional reduced data representation. Patient profiles for each cluster are shown in Table 1. The disease duration, ALSFRS-R, ΔALSFRS-R, BMI, FVC, ΔFVC, the number of patients using EN and NIV significantly differed between the clusters. Cluster 1 exhibited the shortest disease duration (15.0 (10.3) months), with relatively preserved motor neuron function, as indicated by ALSFRS-R score (40.5 (7.3)), BMI (21.6 (4.4) kg/m2), and FVC (87.8% (21.6)). In contrast, clusters 2 and 3 had longer disease durations (51.5 (14.5) months and 45.0 (23.0) months, respectively). Cluster 2 showed preserved ALSFRS-R scores 38.0 (2.5), BMI (22.2 (3.5) kg/m2), and FVC (100.7% (14.0)). Cluster 3 demonstrated advanced symptoms with a low ALSFRS-R score (23.0 (9.0)), low BMI (19.5 (2.6) kg/m2), and low FVC (39.5% (15.1)). Based on these characteristics, three groups were named as follows: “Group 1: early-preserved group” (n = 18), “Group 2: late-preserved group” (n = 10), and “Group 3: late-impaired group” (n = 17). Heart Rate Variability Measurement Table 2 shows the HRV and HR at the Rest phase in the control subjects and overall patients. Overall, patients showed lower HF and higher LF/HF, regardless of normal LF, compared with controls. These differences were particularly evident in Groups 1 and 3. Both groups had lower HF ( p = 0.003 and p = 0.006, respectively) and higher LF/HF ( p = 0.004 and p = 0.01, respectively) than controls.LF in Group 3 was low but not significantly different from the control value. In contrast, Group 2 showed no significant differences in any parameter with the controls, although LF/HF was slightly higher than that in the controls. The HR in the Rest phase showed no differences from the control values in any group. Figure 2 shows the variations in HRV and HR at the Rest, Task, and After phases in the control subjects and overall patients with ALS. In control subjects, HF significantly decreased from Rest to Task (Fig. 2 a, p = 0.028) and increased from Task to After (Fig. 2 a, p < 0.001). In contrast, LF/HF increased from Rest to Task (Fig. 2 c, p = 0.002) and decreased from Task to After (Fig. 2 c, p = 0.0498), although LF showed no significant changes between each phase (Fig. 2 b). HR increased in the Task phase and returned to the baseline value in the After phase (Fig. 2 d). In all patients with ALS, no variations across the phases were observed for HF, LF, and LF/HF (Fig. 2 a-c). There were significant variations only in the HR in the patients (Fig. 2 d). Table 3 compares the ratio of each value at Task/Rest and After/Task. Significant differences in the Task/Rest and After/Task ratios were observed between control subjects and the overall patients in HF ( p < 0.001 and p < 0.001, respectively), LF/HF ( p < 0.001 and p = 0.002, respectively), and HR ( p < 0.001 and p < 0.001, respectively). Figure 3 shows the variations in HRV and HR in each patient group. Among the three groups, only Group 2 showed a significant increase in HF at the After phase, similarly to the control subjects (Fig. 3 a, p = 0.037). For LF and LF/HF, no significant changes were observed in any groups (Fig. 3 b, c), although in Group 2, the LF/HF at the Task phase was not significantly higher than at the Rest phase (Fig. 3 c). As shown in Fig. 3 d, the variations in HR across the phases in each group were minimal compared to those in the control subjects. Table 3 compares the ratio of values at Task/Rest and After/Task in the control group and each patient group. Significant differences in the Task/Rest and After/Task ratios were observed between groups for HF ( p = 0.004 and p < 0.001, respectively), LF/HF ( p = 0.004 and p = 0.021, respectively), and HR ( p < 0.001 and p < 0.001, respectively). Furthermore, the post hoc test using Dunn’s test revealed significant differences in all variables between the control subjects and Group 1 and between the control subjects and Group 3. Discussion This study investigated autonomic function in patients with ALS with varying severity and disease progression. Overall, patients with ALS had lower HF and higher LF/HF ratios than the controls. LF reflects sympathetic and parasympathetic activity, whereas HF reflects parasympathetic activity [ 35 ]. It has been suggested that patients with ALS show a shift in autonomic balance toward sympathetic hyperactivity and parasympathetic hypoactivity, or sympathovagal imbalance [ 11 – 14 ]. The blunted response of LF/HF response during the task in patients might have been caused by ceiling effects due to high LF/HF [ 27 ]. The novel finding of this study is that these autonomic functions differ depending on the severity and progression of ALS. The abnormalities were more prominent in Group 1 (early-preserved group) and Group 3 (late-impaired group) than in Group 2 (late-preserved group). Alterations in autonomic function may be related to the variability in ALS disease progression. Accumulating evidence from previous neurophysiological studies has established that patients with ALS exhibit sympathetic hyperactivity and sympathovagal imbalance. Our findings show that the decreased HF and increased LF/HF ratios are consistent with previous reports [ 13 ]. Although the pathophysiology of these autonomic dysregulations has not been clarified, limbic system abnormalities have been suggested as a probable etiology of sympathetic hyperactivity [ 7 , 10 ]. ALS is a multisystem disorder involving frontotemporal lobes and central sensory and autonomic pathways [ 1 , 7 ]. The brain centers of autonomic regulation, including the insular cortex, cingulate gyrus, hypothalamus, central grey matter, and brainstem autonomic centers, may be involved, at least functionally and even pathologically, in the advanced stages of ALS [ 36 ]. The results at the Rest phase showed abnormal findings in Groups 1 and 3, but not in Group 2. The abnormalities in the overall patients might reflect the results in Groups 1 and 3. While having a short disease duration, Group 1 (the early-preserved group) showed large values of ∆ALSFRS-R, ∆BMI, and ∆FVC, comparable to those in the late-impaired group. This suggests that this group is characterized by rapid disease progression in which autonomic regulatory functions are already affected from the early stages of ALS. Psychological stress and the disease pathophysiology might contribute to sympathetic hyperactivity [ 7 , 17 ]. Group 3 (the late-impaired group) included typical advanced cases of ALS with long disease duration, lower ALSFRS-R, lower BMI, and lower FVC. This group showed markedly lower HF and higher LF/HF at rest and blunted LF/HF and HR responses than controls, similarly to the results in Group 1. This similarity indicates that Group 1 patients may develop clinical characteristics similar to those in Group 3 along with disease progression. Group 2, the late-preserved group, showed different results from the other groups. None of the HRV values at rest in Group 2 showed significant differences from the control values. Furthermore, HF showed a significant increase in response to the end of the task load, and the ratio of values at Task/Rest and After/Task showed smaller differences from the controls compared to Groups 1 and 3. These results indicate that this group exhibited autonomic regulation similar to healthy subjects. Despite the long disease duration, this group maintained better motor and respiratory function than the others. Previous studies reported that patients with ALS and FVC < 50% showed lower HRV compared to those with FVC ≥ 50% [ 18 ]. Considering that HRV is affected by respiratory dysfunction, the preserved respiratory function in Group 2 might have ameliorated autonomic dysfunction. Group 1, the early-preserved group, demonstrated autonomic abnormalities despite relatively preserved motor function, suggesting that dysregulation can emerge at early disease stages. Given their rapid progression, these patients may eventually resemble Group 3, the late-impaired group. In clinical practice, disease-modifying therapies might slow ALS progression, potentially enabling a shift toward the late-preserved phenotype and maintaining autonomic stability. Although parasympathetic dysfunction is known to worsen with disease progression [ 20 ], our findings indicate that autonomic regulation can be compromised even at early stages, while in some patients it remains preserved despite long disease duration. Further longitudinal studies are required to clarify whether early therapeutic interventions can alter HRV abnormalities and improve prognosis. This study had some limitations. First, the sample size was small. Although our sample may not strictly represent the entire ALS population, the use of cluster analysis allowed for the consideration of heterogeneity within the patient group, which is a strength of this study. However, the potential for selection bias and limitations in external validity should be acknowledged, and further validation through large-scale studies is warranted. Second, the task differed between controls and patients. Control subjects performed a random number generation task, whereas patients performed a serial seven test. In this study, we considered both tasks equivalent to mental loads. Third, we did not investigate the psychological symptoms of patients. Psychological stress, anxiety, and irritation may influence HRV [ 23 ], highlighting the need to incorporate such measures into future investigations. Fourth, the age of the controls was lower than that of patients with ALS. Aging has been reported to affect autonomic function. Th analyses in this study were conducted without adjusting for age differences, which may have influenced the results. This study found that autonomic regulatory functions differ depending on the varying severity and progression of ALS. Patients with preserved overall function and BMI, even those with long disease duration, may be less susceptible to autonomic dysfunction. Longitudinal studies are needed to elucidate this possibility. Abbreviations ALS amyotrophic lateral sclerosis ALSFRS-R the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale BMI body mass index EN enteral nutrition FVC forced vital capacity HF high-frequency HR heart rate HRV heart rate variability KNN k-nearest neighbor LF low-frequency LF/HF LF/HF ratio MSNA muscle sympathetic nerve activity NIV non-invasive ventilation OPTICS Ordering Points To Identify the Clustering Structure SSNA skin sympathetic nerve activity TIV tracheostomy and invasive ventilation UMAP Uniform Manifold Approximation and Projection Declarations Declarations Ethics approval and consent to participate This study was approved by the ethics committee of the Tokyo Metropolitan Neurological Hospital (No. R03-019) and the Tokyo Metropolitan Institute of Medical Science (No. 22-19). This study was conducted according to the principles of the Declaration of Helsinki. Informed consent was obtained from all the participants or their representatives included in the study. Consent for publication All patients or their representatives provided written informed consent for publication. Availability of data and materials Data supporting the findings of this study are available upon request from the corresponding authors. The data are not publicly available because they contain information that can compromise the privacy of the research participants. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI. under Grant-in-Aid for Scientific Research [B] 22H03398 and 23K24656 Authors' contributions Y.N. was responsible for the conception and design of the study, data collection, data analysis and interpretation, and drafting of the manuscript. J.S. primarily conducted data collection, data analysis and interpretation, and took the lead in manuscript writing. K.S. and S.F. performed advanced data analyses, created Figures 1–3, contributed to data interpretation, and made substantial revisions to the manuscript. C.M., M.H., and Y.M. contributed to study design and data collection, and also checked and revised the text, figures, and tables for accuracy and clarity. K.B., K.H., and R.M. contributed to study design and data collection, supervised the data analyses, and provided critical input on interpretation. T.Shiba. contributed to study design, conducted comparisons with control data, and performed HRV-related data analyses and interpretation. T.Shimizu. contributed to study design, supervised overall data analyses, provided critical input into data interpretation, and played a major role in manuscript writing and revision. 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Hahsler M, Piekenbrock M, Doran D. dbscan: fast density-based clustering with R. J Stat Softw. 2019;91:1–30. 10.18637/jss.v091.i01 . Melville J. The Uniform Manifold Approximation and Projection (UMAP) Method for Dimensionality Reduction [R package uwot version 0.2.2]. 2024 Apr 21. Accessed 2024 Dec 19. https://CRAN.R-project.org/package=uwot Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Heart rate variability: Standards of measurement, physiological interpretation, and clinical use. Eur Heart J. 1996;17:354–81. 10.1093/oxfordjournals.eurheartj.a014868 . Hayashi K, Mochizuki Y, Takeuchi R, et al. Clinicopathological characteristics of patients with amyotrophic lateral sclerosis resulting in a totally locked-in state (communication Stage V). Acta Neuropathol Commun. 2016;4:1–14. 10.1186/s40478-016-0379-3 . Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files table1.xlsx table2.xlsx table3.xlsx Cite Share Download PDF Status: Published Journal Publication published 01 May, 2026 Read the published version in BMC Neurology → Version 1 posted Editorial decision: Revision requested 22 Dec, 2025 Reviews received at journal 18 Dec, 2025 Reviewers agreed at journal 04 Dec, 2025 Reviews received at journal 03 Dec, 2025 Reviewers agreed at journal 02 Dec, 2025 Reviewers invited by journal 02 Dec, 2025 Editor invited by journal 30 Oct, 2025 Editor assigned by journal 14 Oct, 2025 Submission checks completed at journal 14 Oct, 2025 First submitted to journal 14 Oct, 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. 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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-7862037","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":554523939,"identity":"8fa22883-184d-4019-95ba-e4513ae2017d","order_by":0,"name":"Juri Sawada","email":"","orcid":"","institution":"Tokyo Metropolitan Institute of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Juri","middleName":"","lastName":"Sawada","suffix":""},{"id":554523940,"identity":"d77141d7-b2ae-4de7-927c-4609c12f3a21","order_by":1,"name":"Yuki 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01:56:45","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":112246,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7862037/v1/53022512502c2f0335e82791.html"},{"id":97488990,"identity":"d5f62535-557c-4613-aec3-72a05dc723f8","added_by":"auto","created_at":"2025-12-05 01:56:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52708,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Distribution of clusters detected by Ordering Points To Identify the Clustering Structure (OPTICS) on the two-dimensional reduced representation of the study data.\u003c/p\u003e\n\u003cp\u003e(b) Uniform Manifold Approximation and Projection clusters for the two-dimensional reduced representation of the data annotated by the clusters generated by OPTICS.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7862037/v1/bd0ce36c0bf1433611504710.png"},{"id":97488992,"identity":"4bcb48d5-9c16-4064-816e-d2a8c787ae1f","added_by":"auto","created_at":"2025-12-05 01:56:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93730,"visible":true,"origin":"","legend":"\u003cp\u003eComparisons of heart rate variability (HRV) across the Rest, Task, and After phases in control subjects and overall patients with amyotrophic lateral sclerosis. High-frequency (HF, a), low-frequency (LF, b), LF/HF ratio (c), and heart rate (HR, d) were compared across three-time points (Rest, Task, and After) for each group. Statistical analysis was conducted using the Friedman test to assess within-cluster temporal changes.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7862037/v1/b8065365b6bc735a974bd571.png"},{"id":97669879,"identity":"555f7457-f01e-41ba-9255-a643089b72e7","added_by":"auto","created_at":"2025-12-08 09:29:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":152876,"visible":true,"origin":"","legend":"\u003cp\u003eComparisons of heart rate variability (HRV) across the Rest, Task, and After phases in control subjects and each group. High-frequency (HF, a), low-frequency (LF, b), LF/HF ratio(c), and heart rate (HR, d) were compared across three-time points (Rest, Task, and After) for each group. Statistical analysis was conducted using the Friedman test to assess within-cluster temporal changes.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7862037/v1/7cf1d5ded0578f5696f90c44.png"},{"id":108437548,"identity":"9b9e4304-a82f-4173-a273-1f3a4da20dca","added_by":"auto","created_at":"2026-05-04 15:59:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":524939,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7862037/v1/6035b80a-f20f-4a72-a3a0-555934e62d52.pdf"},{"id":97670638,"identity":"cfefbc18-7ece-4d95-81b7-76ff470e8d1f","added_by":"auto","created_at":"2025-12-08 09:31:04","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13098,"visible":true,"origin":"","legend":"","description":"","filename":"table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7862037/v1/b71bdef5993c4bdcd25c7506.xlsx"},{"id":97670838,"identity":"1bee9dfb-6a5c-4aa5-85e5-43e164876561","added_by":"auto","created_at":"2025-12-08 09:31:24","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12576,"visible":true,"origin":"","legend":"","description":"","filename":"table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7862037/v1/78cc077b43587da288bf61d4.xlsx"},{"id":97670983,"identity":"08e125f9-4ed0-43e0-b66d-f58a4d3c6ee4","added_by":"auto","created_at":"2025-12-08 09:31:40","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13814,"visible":true,"origin":"","legend":"","description":"","filename":"table3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7862037/v1/84a30479262bf315a6ea373a.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cluster analysis of heart rate variability reveals subgroups with preserved and early- impaired autonomic regulation in amyotrophic lateral sclerosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons. However, recent studies have indicated that it may be involved in multisystem degeneration affecting autonomic and sensory neurons [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Non-motor symptoms unrelated to motor neuron impairment are observed in 5%\u0026ndash;80% of patients with ALS [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and significantly reduce their quality of life [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Autonomic symptoms of ALS include cardiovascular, gastrointestinal, voiding, and sudomotor dysfunction [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Urinary and gastrointestinal symptoms are noted in 30% of patients with ALS [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. While these autonomic dysfunctions may appear mild, some patients with tracheostomy and invasive ventilation (TIV) in the advanced stages may present prominent fluctuations of blood pressure and heart rate, known as \u0026ldquo;autonomic storm,\u0026rdquo; which can lead to sudden death [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This condition is caused by central sympathetic hyperactivity and downregulation of peripheral sympathetic receptor function, which may result in circulatory collapse [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, since unstable blood pressure is associated with disease progression [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], autonomic dysfunction is a significant concern in ALS.\u003c/p\u003e\u003cp\u003eRecent studies have reported an imbalance between the sympathetic and parasympathetic systems in ALS [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Assessment of heart rate variability (HRV), a non-invasive measure of autonomic function, revealed that patients with ALS exhibited reduced autonomic activity with sympathetic predominance compared to healthy individuals [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Neurophysiological studies indicated that muscle sympathetic nerve activity (MSNA) and skin sympathetic nerve activity (SSNA) initially increased and then decreased with age and disease duration [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Secondary factors such as psychological stress, long-term ventilatory support, severe muscle atrophy, long-term bedridden state, and repetitive infections may contribute to sympathetic hyperactivity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although these autonomic abnormalities are evident, including sympathetic hyperactivity and concomitant/subsequent blunted vascular responses, the mechanisms and factors underlying these autonomic dysfunctions remain unclear.\u003c/p\u003e\u003cp\u003eFocusing on patients with ALS at the stages before ventilator use, previous studies reported that decreased HRV was associated with decreased lung capacity and increased disease duration [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In contrast, a study found respiratory dysfunction was not associated with decreased parasympathetic activity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Considering the heterogeneous progression of ALS, several additional measures that capture ALS progression must be considered. Although most studies examined autonomic function at rest, evaluating autonomic function in response to tasks may provide a more comprehensive assessment. Understanding the factors associated with autonomic dysregulation in ALS could lead to early and effective interventions before TIV implementation.\u003c/p\u003e\u003cp\u003eThis study examined whether autonomic dysregulation differs across patients with ALS with varying severity and progression using non-invasive HRV measurements. This study aimed to identify factors associated with autonomic dysregulation in ALS by assessing sympathetic and parasympathetic nervous system functions under task conditions.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eParticipants\u003c/h2\u003e\u003cp\u003eWe enrolled 45 patients who visited the Tokyo Metropolitan Neurological Hospital between March 2017 and September 2025 and were diagnosed with sporadic ALS. Although the enrollment of patients was not consecutive, we enrolled patients who provided written informed consent to participate in the study. All patients were diagnosed with ALS according to the revised El Escorial criteria as \u0026ldquo;clinically definite,\u0026rdquo; \u0026ldquo;clinically probable,\u0026rdquo; \u0026ldquo;clinically probable-laboratory supported,\u0026rdquo; or \u0026ldquo;clinically possible\u0026rdquo; ALS [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Patients with a history of alcohol or tobacco abuse, arrhythmias, or peripheral neuropathy were excluded. There were no patients who were taking medications that could affect autonomic functions at the time of investigation. All the participants maintained sufficient cognitive function to meet the task requirements.\u003c/p\u003e\u003cp\u003eThe following clinical characteristics were assessed: sex, age at onset, age at evaluation, height, premorbid weight, body weight at diagnosis, body weight at evaluation, disease duration (months), onset region (bulbar, upper or lower limb), use of enteral nutrition (EN), use of non-invasive ventilation (NIV), the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) at evaluation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and seated forced vital capacity (FVC) at evaluation. Thereafter, body mass index (BMI) was calculated as weight (kg)/height (m)\u003csup\u003e2\u003c/sup\u003e. FVC was expressed as a percentage and calculated as follows: (measured FVC/predicted FVC) \u0026times; 100. ΔALSFRS-R was calculated as follows: (48 ALSFRS-R score at evaluation)/disease duration (months). ΔFVC was calculated as (100 \u0026ndash; measured FVC)/disease duration (months) and ΔBMI as (premorbid BMI \u0026ndash; BMI at evaluation)/disease duration (months).\u003c/p\u003e\u003cp\u003eEleven healthy subjects (median 59.0 (IQR 10.5) years, eight males) served as the control population [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This study was approved by the ethics committee of the Tokyo Metropolitan Neurological Hospital (No. R03-019) and the Tokyo Metropolitan Institute of Medical Science (No. 22\u0026thinsp;\u0026minus;\u0026thinsp;19). This study was performed according to the ethical standards described in the latest version of the Declaration of Helsinki and the Ethical Guidelines for Clinical Research of the Tokyo Metropolitan Neurological Hospital. All participants provided written informed consent before participating in the study.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHeart Rate Variability Measurement\u003c/h3\u003e\n\u003cp\u003eHRV measurements were conducted in the outpatient examination room between 13:00 and 17:00 after routine consultations. Patients lay on a bed, and a quiet environment was set up for HRV measurements in a relaxed state. The three-behavioral-state paradigm consisting of the \u0026lsquo;Rest,\u0026rsquo; \u0026lsquo;Task,\u0026rsquo; and \u0026lsquo;After\u0026rsquo; phases was applied based on previous studies [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. During the Rest phase, patients relaxed on a bed for 120 s. For the Task phase, a 60-second silent Serial Sevens Test was performed in patients\u0026rsquo; minds at their own pace. Finally, during the After phase, patients were instructed to cease the calculation and relax again for 120 s. The Serial Sevens Test, a mental arithmetic task with a high cognitive load [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], was used to assess mental stress in patients [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the present study, each patient performed calculations in mind from \u0026lsquo;100 minus 7,\u0026rsquo; \u0026lsquo;93 minus 7,\u0026rsquo; and so on. We verified task completion by asking the patient the final number after the measurement was completed. Control subjects performed a random number generation task during the Task phase, as previously reported [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Data on control subjects were obtained from previous independent studies conducted by one of the co-authors [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Although the tasks\u0026rsquo; characteristics differed between patients and control subjects in this study, we judged that the two tasks had equivalent loads to mental tasks.\u003c/p\u003e\u003cp\u003eSpectral analysis of the HRV, which is widely used to assess autonomic function, was conducted using an ECG monitor (RF-ECG2; GM3, Tokyo, Japan) attached to the chest. ECG data were recorded on a computer, and R-R intervals were analyzed using the maximum entropy method (MemCalc, GMS, Tokyo, Japan) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This method allows the analysis of short, 30-second data segments [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and was applied to the three-behavioral-state paradigm in this study [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. MemCalc calculated the low-frequency (LF) and high-frequency (HF) components every two seconds by integrating power within frequency ranges of 0.04\u0026ndash;0.15 Hz for LF and 0.15\u0026ndash;0.4 Hz for HF. The heart rate (HR, bpm) was calculated from the R-R intervals [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eWe utilized Uniform Manifold Approximation and Projection (UMAP) and Ordering Points To Identify the Clustering Structure (OPTICS) algorithms to characterize the clinical state of patients with ALS [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Using UMAP, nine variables that reflect the severity and progression of ALS (age, disease duration, ALSFRS-R, FVC, BMI, ΔBMI, and three binary variables: bulbar onset, EN use, and NIV use) were reduced to a two-dimensional space, allowing for visualization of the data structure [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In the dataset used, the FVC values were missing for two people (5.7% of the total); therefore, we used the k-nearest neighbor (KNN) method to fill in the missing values [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. A composite distance metric combining the Euclidean distance for continuous variables and the Hamming distance for binary variables was applied, with the five nearest neighbors specified for each data point and a minimum distance parameter of 0.01. OPTICS analyzes the density structures in the reduced data, identifies high-density regions based on a 5% minimum point threshold, and generates a reachability plot. Clustering structures were visualized using this plot, and distinct clusters were identified using the Density-Based Spatial Clustering of Applications with Noise (DBSCAN) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The DBSCAN distance threshold was determined from the \u0026ldquo;elbow\u0026rdquo; of the KNN distance plot.\u003c/p\u003e\u003cp\u003eAfter assigning the patients to clusters, nine variables were summarized for each cluster. Differences in distributions between clusters were assessed to characterize the disease states. The Shapiro-Wilk test was used to evaluate variable distributions, guiding the choice of statistical methods. The Kruskal-Wallis test was used to compare continuous data between clusters, whereas the chi-squared test was used to analyze categorical data.\u003c/p\u003e\u003cp\u003eWe compared the resting HRV and resting HR values of the following groups to evaluate the differences in autonomic nervous dysfunction between patients with ALS and control subjects: control subjects vs. all patients and control subjects vs. each cluster. The Mann\u0026ndash;Whitney U test was used for comparison. Next, we compared the changes in HRV and HR values in the three phases (Rest, Task, and After) between control subjects, all patients with ALS, and each cluster. We used Friedman\u0026rsquo;s repeated-measures test for this analysis and Nemenyi\u0026rsquo;s test for post-hoc comparisons.\u003c/p\u003e\u003cp\u003eFinally, we compared the changes in HRV and HR values using the ratio of values in the Task phase to the Rest phase (Task/Rest) and the After phase to the Task phase (After/Task). Comparisons between control subjects and all patients were performed using the Mann-Whitney U test. In contrast, comparisons between control subjects and each cluster were performed using the Kruskal\u0026ndash;Wallis test and a post-hoc Dunn\u0026rsquo;s test.\u003c/p\u003e\u003cp\u003eAll statistical analyses were conducted using R software (version 4.4.0) with the Uwot and DBSCAN packages [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], with the significance level set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003ePatient characteristics\u003c/h2\u003e\u003cp\u003eThe clinical characteristics of the patients are summarized in Table\u0026nbsp;1. The median age at examination was 64.0 (IQR 15.0) years. While eleven patients were using NIV, all used it either only at night or intermittently during the day, and none were using it at the time of the examination. Among the 19 patients using EN, 18 used gastrostomy tubes, and one used a nasogastric tube; all patients were in stable condition without pain or wound inflammation following gastrostomy tube placement.\u003c/p\u003e\u003cp\u003eWe identified three subgroups (clusters) among the patients using the OPTICS clustering algorithm. The distribution of clusters is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a). In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b), the clusters detected by OPTICS are visualized by performing UMAP on a two-dimensional reduced data representation. Patient profiles for each cluster are shown in Table\u0026nbsp;1. The disease duration, ALSFRS-R, ΔALSFRS-R, BMI, FVC, ΔFVC, the number of patients using EN and NIV significantly differed between the clusters. Cluster 1 exhibited the shortest disease duration (15.0 (10.3) months), with relatively preserved motor neuron function, as indicated by ALSFRS-R score (40.5 (7.3)), BMI (21.6 (4.4) kg/m2), and FVC (87.8% (21.6)). In contrast, clusters 2 and 3 had longer disease durations (51.5 (14.5) months and 45.0 (23.0) months, respectively). Cluster 2 showed preserved ALSFRS-R scores 38.0 (2.5), BMI (22.2 (3.5) kg/m2), and FVC (100.7% (14.0)). Cluster 3 demonstrated advanced symptoms with a low ALSFRS-R score (23.0 (9.0)), low BMI (19.5 (2.6) kg/m2), and low FVC (39.5% (15.1)). Based on these characteristics, three groups were named as follows: \u0026ldquo;Group 1: early-preserved group\u0026rdquo; (n\u0026thinsp;=\u0026thinsp;18), \u0026ldquo;Group 2: late-preserved group\u0026rdquo; (n\u0026thinsp;=\u0026thinsp;10), and \u0026ldquo;Group 3: late-impaired group\u0026rdquo; (n\u0026thinsp;=\u0026thinsp;17).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eHeart Rate Variability Measurement\u003c/h2\u003e\u003cp\u003eTable\u0026nbsp;2 shows the HRV and HR at the Rest phase in the control subjects and overall patients. Overall, patients showed lower HF and higher LF/HF, regardless of normal LF, compared with controls. These differences were particularly evident in Groups 1 and 3. Both groups had lower HF (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003 and p\u0026thinsp;=\u0026thinsp;0.006, respectively) and higher LF/HF (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01, respectively) than controls.LF in Group 3 was low but not significantly different from the control value. In contrast, Group 2 showed no significant differences in any parameter with the controls, although LF/HF was slightly higher than that in the controls. The HR in the Rest phase showed no differences from the control values in any group.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the variations in HRV and HR at the Rest, Task, and After phases in the control subjects and overall patients with ALS. In control subjects, HF significantly decreased from Rest to Task (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, p\u0026thinsp;=\u0026thinsp;0.028) and increased from Task to After (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, LF/HF increased from Rest to Task (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, p\u0026thinsp;=\u0026thinsp;0.002) and decreased from Task to After (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, p\u0026thinsp;=\u0026thinsp;0.0498), although LF showed no significant changes between each phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). HR increased in the Task phase and returned to the baseline value in the After phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). In all patients with ALS, no variations across the phases were observed for HF, LF, and LF/HF (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). There were significant variations only in the HR in the patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Table\u0026nbsp;3 compares the ratio of each value at Task/Rest and After/Task. Significant differences in the Task/Rest and After/Task ratios were observed between control subjects and the overall patients in HF (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively), LF/HF (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002, respectively), and HR (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the variations in HRV and HR in each patient group. Among the three groups, only Group 2 showed a significant increase in HF at the After phase, similarly to the control subjects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, p\u0026thinsp;=\u0026thinsp;0.037). For LF and LF/HF, no significant changes were observed in any groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c), although in Group 2, the LF/HF at the Task phase was not significantly higher than at the Rest phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, the variations in HR across the phases in each group were minimal compared to those in the control subjects. Table\u0026nbsp;3 compares the ratio of values at Task/Rest and After/Task in the control group and each patient group. Significant differences in the Task/Rest and After/Task ratios were observed between groups for HF (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively), LF/HF (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021, respectively), and HR (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). Furthermore, the post hoc test using Dunn\u0026rsquo;s test revealed significant differences in all variables between the control subjects and Group 1 and between the control subjects and Group 3.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigated autonomic function in patients with ALS with varying severity and disease progression. Overall, patients with ALS had lower HF and higher LF/HF ratios than the controls. LF reflects sympathetic and parasympathetic activity, whereas HF reflects parasympathetic activity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It has been suggested that patients with ALS show a shift in autonomic balance toward sympathetic hyperactivity and parasympathetic hypoactivity, or sympathovagal imbalance [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The blunted response of LF/HF response during the task in patients might have been caused by ceiling effects due to high LF/HF [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The novel finding of this study is that these autonomic functions differ depending on the severity and progression of ALS. The abnormalities were more prominent in Group 1 (early-preserved group) and Group 3 (late-impaired group) than in Group 2 (late-preserved group). Alterations in autonomic function may be related to the variability in ALS disease progression.\u003c/p\u003e\u003cp\u003eAccumulating evidence from previous neurophysiological studies has established that patients with ALS exhibit sympathetic hyperactivity and sympathovagal imbalance. Our findings show that the decreased HF and increased LF/HF ratios are consistent with previous reports [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although the pathophysiology of these autonomic dysregulations has not been clarified, limbic system abnormalities have been suggested as a probable etiology of sympathetic hyperactivity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. ALS is a multisystem disorder involving frontotemporal lobes and central sensory and autonomic pathways [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The brain centers of autonomic regulation, including the insular cortex, cingulate gyrus, hypothalamus, central grey matter, and brainstem autonomic centers, may be involved, at least functionally and even pathologically, in the advanced stages of ALS [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe results at the Rest phase showed abnormal findings in Groups 1 and 3, but not in Group 2. The abnormalities in the overall patients might reflect the results in Groups 1 and 3. While having a short disease duration, Group 1 (the early-preserved group) showed large values of ∆ALSFRS-R, ∆BMI, and ∆FVC, comparable to those in the late-impaired group. This suggests that this group is characterized by rapid disease progression in which autonomic regulatory functions are already affected from the early stages of ALS. Psychological stress and the disease pathophysiology might contribute to sympathetic hyperactivity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Group 3 (the late-impaired group) included typical advanced cases of ALS with long disease duration, lower ALSFRS-R, lower BMI, and lower FVC. This group showed markedly lower HF and higher LF/HF at rest and blunted LF/HF and HR responses than controls, similarly to the results in Group 1. This similarity indicates that Group 1 patients may develop clinical characteristics similar to those in Group 3 along with disease progression.\u003c/p\u003e\u003cp\u003eGroup 2, the late-preserved group, showed different results from the other groups. None of the HRV values at rest in Group 2 showed significant differences from the control values. Furthermore, HF showed a significant increase in response to the end of the task load, and the ratio of values at Task/Rest and After/Task showed smaller differences from the controls compared to Groups 1 and 3. These results indicate that this group exhibited autonomic regulation similar to healthy subjects. Despite the long disease duration, this group maintained better motor and respiratory function than the others. Previous studies reported that patients with ALS and FVC\u0026thinsp;\u0026lt;\u0026thinsp;50% showed lower HRV compared to those with FVC\u0026thinsp;\u0026ge;\u0026thinsp;50% [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Considering that HRV is affected by respiratory dysfunction, the preserved respiratory function in Group 2 might have ameliorated autonomic dysfunction.\u003c/p\u003e\u003cp\u003eGroup 1, the early-preserved group, demonstrated autonomic abnormalities despite relatively preserved motor function, suggesting that dysregulation can emerge at early disease stages. Given their rapid progression, these patients may eventually resemble Group 3, the late-impaired group. In clinical practice, disease-modifying therapies might slow ALS progression, potentially enabling a shift toward the late-preserved phenotype and maintaining autonomic stability. Although parasympathetic dysfunction is known to worsen with disease progression [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], our findings indicate that autonomic regulation can be compromised even at early stages, while in some patients it remains preserved despite long disease duration. Further longitudinal studies are required to clarify whether early therapeutic interventions can alter HRV abnormalities and improve prognosis.\u003c/p\u003e\u003cp\u003eThis study had some limitations. First, the sample size was small. Although our sample may not strictly represent the entire ALS population, the use of cluster analysis allowed for the consideration of heterogeneity within the patient group, which is a strength of this study. However, the potential for selection bias and limitations in external validity should be acknowledged, and further validation through large-scale studies is warranted. Second, the task differed between controls and patients. Control subjects performed a random number generation task, whereas patients performed a serial seven test. In this study, we considered both tasks equivalent to mental loads. Third, we did not investigate the psychological symptoms of patients. Psychological stress, anxiety, and irritation may influence HRV [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], highlighting the need to incorporate such measures into future investigations. Fourth, the age of the controls was lower than that of patients with ALS. Aging has been reported to affect autonomic function. Th analyses in this study were conducted without adjusting for age differences, which may have influenced the results.\u003c/p\u003e\u003cp\u003eThis study found that autonomic regulatory functions differ depending on the varying severity and progression of ALS. Patients with preserved overall function and BMI, even those with long disease duration, may be less susceptible to autonomic dysfunction. Longitudinal studies are needed to elucidate this possibility.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eALS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eamyotrophic lateral sclerosis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eALSFRS-R\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ethe Revised Amyotrophic Lateral Sclerosis Functional Rating Scale\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBMI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ebody mass index\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eenteral nutrition\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFVC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eforced vital capacity\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehigh-frequency\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eheart rate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHRV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eheart rate variability\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKNN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ek-nearest neighbor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003elow-frequency\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLF/HF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLF/HF ratio\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMSNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emuscle sympathetic nerve activity\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNIV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003enon-invasive ventilation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOPTICS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOrdering Points To Identify the Clustering Structure\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSSNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eskin sympathetic nerve activity\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTIV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etracheostomy and invasive ventilation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eUMAP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eUniform Manifold Approximation and Projection\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThis study was approved by the ethics committee of the Tokyo Metropolitan Neurological Hospital (No. R03-019) and the Tokyo Metropolitan Institute of Medical Science (No. 22-19). This study was conducted according to the principles of the Declaration of Helsinki. Informed consent was obtained from all the participants or their representatives included in the study.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eAll patients or their representatives provided written informed consent for publication.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are available upon request from the corresponding authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data are not publicly available because they contain information that can compromise the privacy of the research participants.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI.\u003c/p\u003e\n\u003cp\u003eunder Grant-in-Aid for Scientific Research [B] 22H03398 and 23K24656\u003c/p\u003e\n\u003cp\u003eAuthors' contributions\u003c/p\u003e\n\u003cp\u003eY.N. was responsible for the conception and design of the study, data collection, data analysis and interpretation, and drafting of the manuscript. J.S. primarily conducted data collection, data analysis and interpretation, and took the lead in manuscript writing. K.S. and S.F. performed advanced data analyses, created Figures 1–3, contributed to data interpretation, and made substantial revisions to the manuscript. C.M., M.H., and Y.M. contributed to study design and data collection, and also checked and revised the text, figures, and tables for accuracy and clarity. K.B., K.H., and R.M. contributed to study design and data collection, supervised the data analyses, and provided critical input on interpretation. T.Shiba. contributed to study design, conducted comparisons with control data, and performed HRV-related data analyses and interpretation. T.Shimizu. contributed to study design, supervised overall data analyses, provided critical input into data interpretation, and played a major role in manuscript writing and revision. All authors contributed to the critical revision of the work and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcknowledgment\u003c/p\u003e\n\u003cp\u003eWe would like to thank all patients who participated in this study. We also sincerely appreciate the support of the healthcare professionals involved.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFeldman EL, Goutman SA, Petri S, et al. Amyotrophic lateral sclerosis. Lancet. 2022;400(10360):1363\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0140-6736(22)01272-7\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(22)01272-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRubio MA, Herrando-Grabulosa M, Navarro X. Sensory involvement in amyotrophic lateral sclerosis. 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Acta Neuropathol Commun. 2016;4:1\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s40478-016-0379-3\u003c/span\u003e\u003cspan address=\"10.1186/s40478-016-0379-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\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":"bmc-neurology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nurl","sideBox":"Learn more about [BMC Neurology](http://bmcneurol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nurl","title":"BMC Neurology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"amyotrophic lateral sclerosis, heart rate variability, autonomic function, sympathetic hyperactivity, sympathovagal imbalance","lastPublishedDoi":"10.21203/rs.3.rs-7862037/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7862037/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003ePatients with amyotrophic lateral sclerosis (ALS) occasionally exhibit autonomic nervous system dysregulation. We examined whether autonomic regulation differed across patients with ALS with varying severity and progression.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eA total of 45 patients with ALS were enrolled and classified into three subgroups using cluster analysis. Heart rate variability was assessed using the maximum entropy method. The low-frequency (LF) and high-frequency (HF) components, LF/HF ratio (LF/HF), and heart rate (HR) were measured. Temporal changes in each parameter during rest, mental tasks, and post-task rest were evaluated. The values for all patients and subgroups were compared with those of 11 healthy control subjects. Between-group differences were evaluated at rest and using the Task/Rest and After/Task ratios, and within-group changes across the three phases were also analyzed, with non-parametric statistical tests applied and significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eCluster analysis classified the patients into three groups: \u0026ldquo;Group 1: early-preserved group\u0026rdquo;, \u0026ldquo;Group 2: late-preserved group\u0026rdquo;, and \u0026ldquo;Group 3: late-impaired group\u0026rdquo;. Overall, the patients showed lower HF and higher LF/HF at rest than the controls, indicating parasympathetic hypoactivity and sympathetic predominance. Abnormalities were more prominent in Groups 1 and 3 than in Group 2. The former two groups showed blunted HF, LF/HF and HR responses during the tasks. The late-preserved group showed no difference in the Task/Rest ratios of HF, LF/HF and HR compared with the controls.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eAutonomic regulatory functions differ depending on the severity and progression of ALS. Autonomic dysregulation in ALS may be associated with a decline in motor function. The presence of HRV abnormalities in early-preserved patients suggests that autonomic dysfunction may precede overt motor decline in rapidly progressing cases. This supports the hypothesis that autonomic dysregulation is not merely a late complication but a fundamental component of ALS pathophysiology. Recognizing HRV abnormalities from early stages may help identify patients at risk of faster progression and guide timely interventions. Future longitudinal studies are needed to confirm whether disease-modifying therapies can alter HRV trajectories and improve prognosis.\u003c/p\u003e","manuscriptTitle":"Cluster analysis of heart rate variability reveals subgroups with preserved and early- impaired autonomic regulation in amyotrophic lateral sclerosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-05 01:56:40","doi":"10.21203/rs.3.rs-7862037/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-22T05:08:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-18T19:09:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277813549715951006420684063293562475903","date":"2025-12-04T20:48:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T08:20:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"341866624427062869240400110174523532","date":"2025-12-02T14:40:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T12:35:53+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-30T18:34:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-15T02:17:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-15T02:17:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Neurology","date":"2025-10-14T21:41:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-neurology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nurl","sideBox":"Learn more about [BMC Neurology](http://bmcneurol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nurl","title":"BMC Neurology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"09994be6-da3e-4f3b-a7b7-4b46584a3a04","owner":[],"postedDate":"December 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T15:58:50+00:00","versionOfRecord":{"articleIdentity":"rs-7862037","link":"https://doi.org/10.1186/s12883-026-04901-w","journal":{"identity":"bmc-neurology","isVorOnly":false,"title":"BMC Neurology"},"publishedOn":"2026-05-01 15:57:01","publishedOnDateReadable":"May 1st, 2026"},"versionCreatedAt":"2025-12-05 01:56:40","video":"","vorDoi":"10.1186/s12883-026-04901-w","vorDoiUrl":"https://doi.org/10.1186/s12883-026-04901-w","workflowStages":[]},"version":"v1","identity":"rs-7862037","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7862037","identity":"rs-7862037","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}