Differential Effects of Sex and Age on Daily and Infradian Rhythms of Mice Running Title: Sex and Age Differences in Mouse Rhythms

Intrinsic biological rhythms regulate key physiological and behavioural processes, yet the influence of sex and age on these rhythms is not fully understood. We comprehensively examined 24-hour (circadian) and >24-hour (infradian; 5-day and 10-day) rhythms in wheel-running and ingestive behaviours in single-housed young and middle-aged male and female mice. Circadian analysis revealed that middle-aged mice, particularly females, exhibited more precise daily rhythms and shifted a greater proportion of activity and feeding to the lights-on phase compared to young female mice. Middle-aged animals also ran for longer durations per day, suggesting age-related changes in activity regulation. Analysis of infradian rhythms further highlighted sex- and age-specific differences. Young female mice displayed robust 5-day rhythms in wheel-running activity, which were absent in middle-aged females. In contrast, few males (young or middle-aged) showed significant 5-day rhythms. Ten-day rhythms were most prominent in male mice, while females rarely expressed this periodicity. Physiologically, middle-aged mice lost more body weight in response to single housing, with middle-aged females being most affected. Interactions among behavioural rhythms in females also showed greater complexity, which increased with age. These findings reveal distinct sex- and age-dependent patterns in circadian and infradian rhythms as well as in physiological responses to isolation. Our work highlights the need to account for sex and age in chronobiological research, with broader implications for understanding vulnerability to age-related metabolic and behavioural disorders.