Minding your confounding variables: the effects of environmental isolation on wheel running behaviors in mice

INTRODUCTION: Physical inactivity is a leading cause of disparity in health and wellness in both human and animal subjects. Providing rodents with a running wheel in the home cage environment allows researchers to assess this complex behavior under various conditions. The use of such methods is a common model of physical activity and inactivity patterns. The effectiveness of a research model to mimic natural populations and responses requires well-structured research designs and special attention to not only experimental methods but also to external environmental conditions. Many animal housing facilities regulate environmental conditions such as light cycles, room temperature, room humidity, and animal husbandry activities. Despite these mechanisms aimed at minimizing confounding variability other potential confounding variables stem from the built environment that are seemingly unavoidable. Room paint color, luminosity levels at various points along the caging rack, and extraneous noise from classrooms, lab spaces, or hallways outside animal husbandry rooms represent potential behavioral effectors. The purpose of this study is to evaluate the effectiveness of exclusionary enclosures on wheel running behaviors in female and male C57BL/6J mice. It is hypothesized that wheel running patterns will be higher when mice are housed in enclosures that account for additional environmental effectors including external sound reduction, equalized luminosity at cage level, and wall coloration. METHODS: Female (n=10) and male (n=9) C57BL/6J mice were individually housed with a solid surface running wheel (circumference = 450 mm) and acclimated to running wheel use for ten days. Following acclimation, mice were evaluated for wheel running distance, duration, and speed under both open and environmentally isolated conditions using a randomized block strategy in which half the mice were in open conditions first while the other half were housed in environmentally isolated conditions first. Environmental exclusion was accomplished with boxes made from neutrally colored sound-reducing foam insulation and were wired with LED lights to standardize illumination to the lowest possible variation at the level of the cage. Wheel running indices were measured by a turn-by-turn running wheel monitoring system for seven days per experimental phase. All mice were provided ad libitum access to food (Teklad Global 18% Protein Rodent Diet) and water throughout the study. Animal husbandry activities were standardized and minimized as much as ethically possible to ensure the lowest possible levels of human/rodent interactions. Both the open and environmentally isolated conditions experienced 12-hour light:12-hour dark cycles. Sex-biased paired Student’s t-tests were used to assess statistical significance for the wheel running indices. RESULTS: Wheel running distance (females; p=0.016 | males; p=0.020) and duration (females; p=0.001 | males; p=0.006) increased in environmentally isolated conditions. Wheel running speed (females; p< 0.001 | males; p=0.015) decreased in in the isolated condition. CONCLUSION: Overall, both female and male mice ran more but at lower speeds when provided environmentally excluded conditions, indicating lower levels of stress and a more precise and natural wheel running behavioral response. Support or funding information This work was supported by intramural grant funding from Union University and the Union University Department of Biology. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.