Effect of Equine Flysheets on Fly Avoidance Behaviors, Body Temperature, Stress Level, and Heart Rate

Effect of Equine Flysheets on Fly Avoidance Behaviors, Body Temperature, Stress Level, and Heart Rate | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 8 January 2026 V1 Latest version Share on Effect of Equine Flysheets on Fly Avoidance Behaviors, Body Temperature, Stress Level, and Heart Rate Authors : Meggan Graves 0000-0002-2559-4402 [email protected] , Xiaocun Sun , and Kellie Fecteau Authors Info & Affiliations https://doi.org/10.22541/au.176790441.19201010/v1 140 views 67 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: Flysheet use is common without any published scientific evidence to justify their use. Objectives: To demonstrate the effectiveness of flysheets at reducing fly avoidance behaviors and measure changes in body temperature, heart rate, or cortisol levels. Study design: 10-week alternating crossover design Methods: 6 horses were divided into two groups. Flysheets were alternated between groups weekly for 10 weeks. Fly avoidance behaviors were recorded for 30-minutes once weekly. Cortisol samples were collected before flysheet application or removal, and again 10 minutes later. Star-Oddi data loggers recorded heartrate and temperature every 15 minutes throughout the study period. Results: Flysheet use significantly reduced fly avoidance behaviors. Mean head tosses decreased from 12.17 (95% CI: 17.58–6.76) to 2.33 (95% CI: 0.64–4.02) (p<0.0001), and tail swishing fell from 214.67 (95% CI: 131.06-298.28) to 52.54 (95% CI: 11.17-93.92) (p0.05). Body temperatures differed by group: Group A had higher mean temperature with flysheets ON (35.85°C, 95% CI: 35.77–35.93) vs. OFF (35.53°C, 95% CI: 35.44–35.62, p<0.0001), whereas Group B had lower mean temperature with flysheets ON (36.42°C, 95% CI: 36.34–36.51) vs. OFF (36.70°C, 95% CI: 36.61–36.79, p<0.0001). Heart rate was significantly higher with flysheets ON (+6.3 bpm, 95% CI: +4.9 to +7.7, p<0.0001). Main limitations: Sample size was small, and one horse was removed from the study due to an unrelated condition. Star-Oddi devices were rejected by two horses creating gaps in data collection. Conclusions: Flysheets effectively reduced insect avoidance behaviors but were associated with increased heart rate and group-dependent effects on body temperature, suggesting a potential thermal or physiologic cost. Flysheet use may improve horse comfort during insect season, but heat load and physiologic stress should be monitored. Effect of Equine Flysheets on Fly Avoidance Behaviors, Body Temperature, Stress Level, and Heart Rate Background: Flysheet use is common without any published scientific evidence to justify their use. Objectives: To demonstrate the effectiveness of flysheets at reducing fly avoidance behaviors and measure changes in body temperature, heart rate, or cortisol levels. Study design: 10-week alternating crossover design Methods: 6 horses were divided into two groups. Flysheets were alternated between groups weekly for 10 weeks. Fly avoidance behaviors were recorded for 30-minutes once weekly. Cortisol samples were collected before flysheet application or removal, and again 10 minutes later. Star-Oddi data loggers recorded heartrate and temperature every 15 minutes throughout the study period. Results: Flysheet use significantly reduced fly avoidance behaviors. Mean head tosses decreased from 12.17 (95% CI: 17.58–6.76) to 2.33 (95% CI: 0.64–4.02) (p<0.0001), and tail swishing fell from 214.67 (95% CI: 131.06-298.28) to 52.54 (95% CI: 11.17-93.92) (p0.05). Body temperatures differed by group: Group A had higher mean temperature with flysheets ON (35.85°C, 95% CI: 35.77–35.93) vs. OFF (35.53°C, 95% CI: 35.44–35.62, p<0.0001), whereas Group B had lower mean temperature with flysheets ON (36.42°C, 95% CI: 36.34–36.51) vs. OFF (36.70°C, 95% CI: 36.61–36.79, p<0.0001). Heart rate was significantly higher with flysheets ON (+6.3 bpm, 95% CI: +4.9 to +7.7, p<0.0001). Main limitations: Sample size was small, and one horse was removed from the study due to an unrelated condition. Star-Oddi devices were rejected by two horses creating gaps in data collection. Conclusions: Flysheets effectively reduced insect avoidance behaviors but were associated with increased heart rate and group-dependent effects on body temperature, suggesting a potential thermal or physiologic cost. Flysheet use may improve horse comfort during insect season, but heat load and physiologic stress should be monitored. 1 Introduction Insect hypersensitivity and fly avoidance behavior is a common occurrence in horses during warmer months. Many horses experience a wide range of behavioral changes such as head shaking, tail swishing and stomping. A subset of these horses may also experience an inflammatory reaction to the insect bites, commonly referred to as sweet itch. In order to improve a horse’s comfort, as well as decrease the incidence of hypersensitivity reactions, owners utilize fly protection techniques such as fly spray, masks and sheets, as well as boots or leggings. These same products have also been used to maintain coat quality for cosmetic purposes. Flysheets are designed to cover the axillary body. Many claim to be breathable and ultraviolet (UV) protective. Horses primarily wear flysheets during peak insect season which tends to be the warmer months (spring through fall). It has been well established that horses thermoregulate through evaporative cooling so the ability to sweat is vital in high temperatures. 1-3 To the authors’ knowledge, no studies have been performed to assess the effect of polyester flysheets on physical exam parameters such as temperature and heart rate, although cotton rugs have been evaluated for effects on thermoregulation. 4 Previous behavioral studies have demonstrated a preference by horses for winter blanket wear in cold, windy or rainy weather. 5,6 However, blankets can cause pressure at the withers leading to discomfort and sores indicating a justification of benefit prior to usage. 7 Older horses prone to hypothermia have been noted to have improved stride length and surface body temperatures from the use of winter blanketing confirming benefits in those situations. 8 Additionally, winter blanketing has been demonstrated to conserve energy resulting in decreased feed intake necessary to maintain body weight and body condition, improving overall feed efficiency. 9 Unlike winter blanketing, minimal studies have been performed to support the efficacy of flysheet usage, although one study did show a reduction in feeding rate of Culicoides . 10 It is difficult to determine the benefits and potential deleterious effects of flysheets without investigating behavior and certain physical exam parameters of the horse as this study aims to do. We hypothesize that flysheets will decrease measured activity levels such as fly avoidance behaviors due to reduced agitation of insects but increase parameters of stress including heart rate and blood cortisol levels. Additionally, we hypothesize that the addition of a flysheet will result in elevated body temperatures in summer months. 2 Materials and Methods All animal procedures were approved by the {masked for review} Institutional Animal Care and Use Committee (#2840-0521). 2.1. Animals This study involved six light breed healthy horses from a teaching herd. Horses ranged in age from 12-15 years old (average 13.75 yrs. +/- 2 years). Four mares and two geldings were utilized. Body condition scores were recorded weekly, all horses were within a 5-6 on a nine-point scale throughout the study. They resumed their typical teaching schedule and management style throughout the duration of the study. The horses were housed in a pasture with a run-in shed for shelter and had access to grass and mixed grass round bale hay. The horses were randomly assigned to two groups (A and B) in an alternating crossover design. Each group contained 3 horses. Flysheets were alternated between groups on a weekly basis for the duration of the study (10 weeks) from August to October. Beginning on day 10, Group A horses wore flysheets while Group B horses did not, with the groups alternating conditions through Day 66. This design allowed each horse to serve as its own control, reducing variability and strengthening comparisons between treatments, with an attempt to account for weather differences. All horses were naïve to flysheets as this is not a normal management practice of the horses enrolled in the study. One horse in Group B was removed early from the study due to circumstances unrelated to the research protocol or study objectives and no data from that horse was utilized. 2.2. Flysheets The flysheets used were Amigo Mio Combo Flysheets (Fig. 1). The sheet is made of a polyester mesh that claims to be breathable and 60% UV protective. It is secured to the horse using Velcro straps under the neck piece, two chest claps, two straps that cross under the abdomen, and cross surcingles. Each horse was properly fitted based on manufacturer guidelines. 2.3 Observations Each horse was individually monitored by the same observer for 30 minutes once weekly throughout the duration of the study. Each monitoring session was held at 7 AM. The behaviors recorded included tail swishing, head tossing, stomping (front or hindlimb), as well as a change in gait (defined as walk, trot, canter). Descriptions of fly avoidance behaviors are found in Table 1 and have been adapted from previous equine and bovine studies. 11,12 2.4 Cortisol Blood samples were collected in red top serum tubes (BD Vacutainer ® ) using a 21g BD Vacutainer™ blood collection needle after the weekly observation period around 7:30 AM. An initial sample was taken, then the flysheets were either removed or applied, depending on the group, and a second cortisol sample was taken 10 minutes later. Samples were immediately stored at 4ºC until centrifuged for 10 minutes at 1500 g. Serum was transferred by pipette to cryovials for storage at -20ºC until analyzed. Serum cortisol concentrations were analyzed using a chemiluminescent immunoassay (Immulite 2000; Siemens). To assess the analytical performance of the immunoassay, within-run precision and within-laboratory precision were determined in pooled serum from 3 horses. Five replicates each day for 3 days were used to determine within-run precision (3.1%) and within-laboratory precision (8.0%). The pooled sample was stored refrigerated between analyses. A spike-and-recovery test was performed by adding assay control material (Lyphochek immunoassay plus control; Biorad) at 1:20 to serum from each of the 3 horses used in the pool. Percent recovery was calculated by dividing the observed value by the expected value and multiplying by 100%. Recovery values were 96.3%, 96.9%, and 131.0%. 2.5 Body Temperature and Heart Rate: Star-Oddi A microsensor (Star-Oddi DST milli or centi-HRT ACT, Gardabaer, Iceland) (Fig. 2) inserted subcutaneously was used to measure body temperature (ºC) and heart rate (beats per minute). Each parameter was recorded every 15 minutes by the microsensor throughout the duration of the study. Heart rate was determined using the microsensor’s ability to interpret the myocardial electric activity in the form of an ECG. The DST milli-HRT ACT is a cylindrical device with dimensions of 13mm x 39.5mm and weighing 12 grams; the DST centri-HRT ACT is 46mm x 15mm weighing 19g. All sensors were sterilized using a 12-hour gas sterilizer and propylene gas. For placement of the Star-Oddi microsensor, a 10 by 10 cm area was clipped and aseptically prepared over the left caudal axillary region. A subcutaneous block with 5-7 mL of 2% lidocaine was performed and a 5 cm diagonal incision through the skin and subcutaneous tissue was made to place the microsensor. The incision was closed with 1 monofilament absorbable PDS II (polydioxanone) suture (Ethicon, Johnson & Johnson, New Brunswick, NJ, United States) using a series of cruciate sutures and sprayed with an aerosol bandage (Neogen®Vet AluSpray® Aluminum Powder). The sutures were removed two weeks later. Data loggers were retrieved at the completion of the study, as all data is stored internally. 2.6 Weather Daily ambient temperatures were obtained from an official weather station within 0.5 mile from the study site. Weather data was recorded at hourly intervals for the duration of the study, along with daily averages. The ambient temperature (ºC) and total amount of rain (mm) were utilized in the study analysis. 3 Results 3.1 Observations Group A was never observed during a rain event when flysheets were ON, and the opposite is true for Group B, making comparisons during rain events unreliable. All observation results are therefore reported for non-rain events only. Horses showed significant reduction in fly avoidance behaviors when flysheets were ON (Table 2). No trotting or cantering was observed during the 30-minute sessions in either group during the study; however, walking was observed. Flysheets did not have a statistically significant effect on walking (p>0.05). Head tossing was greatly reduced in both group A and B when flysheets were ON (p<.0001). During non-rain events, average head tossing for all horses was less when flysheets were ON versus OFF, 2.33 and 12.17 respectively, with a reduction of 80.85% (p<0.0001). Tail swishing was greatly reduced by flysheet wear (p0.05). 3.2 Cortisol Mean cortisol levels were higher for both baseline and 10-minute post flysheet application or removal time points in horses wearing flysheets (Fig. 3). Mean baseline cortisol levels for horses not wearing flysheets was the lowest of all measured (2.92, 95% CI: 2.35-3.48). There was no reduction in cortisol levels over time when comparing baseline early in the project (Day 10) and baseline late in the project (Day 59) (p>0.05) 3.3 Body Temperature A total of 20,911 recordings of temperature measured in degrees Celsius were analyzed. Group A and Group B had minimum/maximum temperature recordings of 32.25/38.04 and 30.81/37.90, respectively, when flysheets were ON. Group A and Group B had minimum/maximum temperature recordings of 31.00/ 39.21 and 32.52/38.89, respectively, when flysheets were OFF. When evaluating least square means, there was no difference in temperature between Groups A and B when flysheets were ON (p>0.05); however, there was a difference when flysheets were OFF (p=.0003). When comparing differences of least square means for Group A horses with flysheets ON and OFF, a statistical difference was noted (p<.0001). The same was observed for Group B horses (p<.0001). For Group A horses, mean body temperatures were lower when flysheets were OFF (35.5348ºC) than when flysheets were ON (35.8520ºC). For Group B horses, mean body temperatures were lower when flysheets were ON (36.4248ºC) than when flysheets were OFF (36.6952ºC). 3.4 Heartrate A total of 18,657 heartrate recordings were analyzed. Mean heartrates were higher among horses wearing flysheets than those not, 49.27 (95% CI: 48.83-49.70) vs. 46.91 (95% CI: 46.53-47.28) bpm respectively (p<.0001) (Fig 4). 3.5 Weather The average ambient temperature during the study period was 21.8175ºC with an average minimum temperature of 17.5565ºC and average maximum temperature of 28.3355ºC. The total rainfall during the study period was 731.318mm. The average maximum relative humidity during the study period was 96.63%. There was no statistical difference in mm of rain (p=0.5), maximum ambient temperature (p>0.05), average ambient daily temperature (p=0.5), or maximum relative humidity (p=0.9) between Group A and B when flysheets were ON. There was also no statistical difference in mm of rain (p=0.5), maximum ambient temperature (p=0.2), average ambient daily temperature (p=0.5), or maximum relative humidity (p=0.9) between Group A and B when flysheets were OFF. When flysheets were ON, Group A had a mean of 14.57mm of rain, and Group B had a mean of 12.24mm of rain. The mean maximum ambient temperature when flysheets were ON for both groups was 28.67ºC, with the mean average ambient temperature being 22.19ºC. The mean maximum relative humidity for both groups when flysheets were ON was 96.6%. 3.6 Data Analysis The effects of flysheet ON/OFF condition, group, time and day on cortisol, observation items, body temperature, as well as heart rate were analyzed using mixed model analysis for repeated measures respectively. The difference between groups on weather data was analyzed using two-sample t-test. Rank data transformation was applied because the diagnostic analysis on residuals exhibited non-normality and unequal variance. Multiple comparisons were conducted with Tukey’s adjustment. Statistical significance was identified at p<0.05. Data were presented as mean and 95% CI. All analyses were conducted in SAS9.4 TS1M8 for Windows 64x (SAS Institute Inc., Cary, NC, USA). 4 Discussion This study aimed to investigate the effects of flysheets on horses by observing their behavior, cortisol levels, body temperature, and heartrate under uncontrolled conditions. Despite the conditions being natural, uncontrolled environments, the lack of significant difference between groups in ambient temperature, rainfall, and relative humidity during different phases of sheet application indicate that external environmental variables were well-controlled throughout the study to allow comparison of the groups. The results of this study highlight significant changes in fly avoidance behaviors and physiological responses when flysheets are used. The primary behavioral outcome noted was a significant reduction in fly avoidance behaviors with the use of flysheets, specifically head tossing and tail swishing. There was no significant reduction in stomping behavior while wearing flysheets, so the addition of leggings may be necessary if limbs seem to be a target area of fly burdens. No statistically significant effect of flysheets on walking was observed, indicating that general movement patterns were not restricted by the sheets and general mobility is not impeded. Cortisol measurements revealed interesting insights into potential stress levels associated with the application and wearing of flysheets. Horses exhibited higher cortisol levels both at baseline and post-application stages when wearing flysheets compared to those not wearing flysheets. This suggests that while the physical discomfort caused by flies is diminished, there may be an initial stress response related to the sensation or restriction caused by the sheets themselves. Additionally, elevated heartrates could indicate a mild stress response or increased metabolic activity associated with adaptation to the sheet. The mixed results of body temperature indicate some thermal impact associated with wearing a flysheet. It is difficult to explain why body temperatures for Group A were slightly higher with sheets ON and not for Group B (body temperatures were lower with sheets ON versus OFF for Group B horses), especially given that weather and environmental factors such as ambient temperature, rainfall, and relative humidity did not differ significantly during the periods of flysheet wear between groups; however, some horses may have sought shade when others did not, as this was not controlled. These results suggest additional factors beyond environmental conditions may influence the relationship between flysheet use and body temperature regulation in horses. Potential explanations could include individual variability in thermoregulation, differences in coat thickness or color, or behavioral responses to wearing flysheets. However, the alternating design of the study complicates interpretation, as it is unclear whether these differences are due to the flysheets themselves or other unmeasured variables. Further research is warranted to explore the mechanisms underlying these observations and how flysheets impact equine thermoregulation and whether their effects vary depending on individual horse characteristics or external factors. In summary, this study provides evidence supporting the effectiveness of flysheets in reducing certain nuisance behaviors like head tossing and tail swishing among horses during non-rain events without significantly restricting movement such as walking. However, physiological data suggest a nuanced picture where initial stress responses indicated by elevated cortisol levels and heartrates might accompany these benefits—an aspect warranting further exploration. Given these horses were naïve to flysheet wear, future work could evaluate acclimation time periods for management practices such as blankets and flysheets with a focus on improving design features to minimize any potential stressors while maximizing their protective benefits. 5 Conclusion Flysheets effectively reduced insect avoidance behaviors but were associated with increased heart rate and group-dependent effects on body temperature, suggesting a potential thermal or physiologic cost. Flysheet use may improve horse comfort during insect season, but heat load and physiologic stress should be monitored, especially in warm weather. Figure 1: Horse pictured wearing an Amigo Mio Combo Flysheet and fitted according to manufacturer guidelines. Figure 2: Star-Oddi DST milli and centi-HRT ACT microsensors utilized to measure and record heartrate and temperature following subcutaneous insertion in the left caudal axillary region of adult horses. Figure 3: Mean cortisol levels (µg/dL) for adult horses with and without flysheets at a baseline time point and 10-minutes following removal or application of flysheets. Figure 4: Mean heartrates of horses with and without flysheets. References 1. Verdegaal ELJMM, Howarth GS, McWhorter TJ, et al. Thermoregulation during Field Exercise in Horses Using Skin Temperature Monitoring. Animals (Basel) 2023;14:136.2. Hodgson DR, Davis RE, McConaghy FF. Thermoregulation in the horse in response to exercise. British veterinary journal 1994;150:219-235.3. Kang H, Zsoldos RR, Sole-Guitart A, et al. Heat stress in horses: a literature review. International journal of biometeorology 2023;67:957-973.4. Padalino B, Loy J, Hawson L, et al. Effects of a light-colored cotton rug use on horse thermoregulation and behavior indicators of stress. Journal of veterinary behavior 2019;29:134-139.5. Meisfjord Jørgensen GH, Mejdell CM, Bøe KE. The effect of blankets on horse behaviour and preference for shelter in Nordic winter conditions. Applied animal behaviour science 2019;218:104822.6. Mejdell CM, Jørgensen GHM, Buvik T, et al. The effect of weather conditions on the preference in horses for wearing blankets. Applied animal behaviour science 2019;212:52-57.7. Clayton HM, Kaiser LJ, Nauwelaerts S. Pressure on the horse’s withers with three styles of blanket. The veterinary journal (1997) 2010;184:52-55.8. Janczarek I, Wiśniewska M, Wnuk-Pawlak E, et al. Effects of horse blankets on the physiological and motion parameters of geriatric horses. Journal of veterinary behavior 2020;38:32-37.9. DeBoer M, Konop A, Fisher B, et al. Dry Matter Intake, Body Weight, and Body Condition Scores of Blanketed and Nonblanketed Horses in the Upper Midwest. Journal of equine veterinary science 2020;94:103239-103239.10. Sloet van Oluitenborgh-Oosterbaan MM. The effect of insect blankets on the feeding rate of Culicoidesspecies in horses in the Netherlands. Journal of veterinary internal medicine 2012;26:427.11. Mullens BA, Lii KS, Mao Y, et al. Behavioural responses of dairy cattle to the stable fly, Stomoxys calcitrans, in an open field environment. Medical and veterinary entomology 2006;20:122-137.12. Mottet RS, Moon RD, Hathaway MR, et al. Effectiveness of Stable Fly Protectants on Adult Horses. Journal of equine veterinary science 2018;69:11-15. BEHAVIOR † DEFINITION Tail Swish Tail moved to either side or up and down from the center line. Stomping Front or hind limb lifted and forcefully returned to the ground. Head Toss Head thrown to either side of the body or down to the chest or forelimbs. † Behaviors and definitions have been adapted from Mullens et al. and Muttet et al. Table 2: Average number and 95% CI of fly avoidance behaviors observed for each group of adult horses when flysheets were either on or off. Flysheet status Behavior observation OFF ON Head tossing 12.17 (95%CI:6.51-17.82) 2.33 (95%CI:0.55-4.11) Stomping 31.4 (95%CI:15.47-47.33) 19.71 (95%CI:6.85-32.57) Tail swishing 214.67 (95%CI:131.06-298.28) 52.54 (95%CI:11.17-93.92) Walking 8.03 (95%CI:4.87-11.19) 4.88 (95%CI:3.16-6.59) Information & Authors Information Version history V1 Version 1 08 January 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Meggan Graves 0000-0002-2559-4402 [email protected] The University of Tennessee Knoxville College of Veterinary Medicine View all articles by this author Xiaocun Sun The University of Tennessee Knoxville View all articles by this author Kellie Fecteau The University of Tennessee Knoxville Department of Biomedical & Diagnostic Sciences View all articles by this author Metrics & Citations Metrics Article Usage 140 views 67 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Meggan Graves, Xiaocun Sun, Kellie Fecteau. Effect of Equine Flysheets on Fly Avoidance Behaviors, Body Temperature, Stress Level, and Heart Rate. Authorea . 08 January 2026. DOI: https://doi.org/10.22541/au.176790441.19201010/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. 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