Ecological specialization is suggested to impede the ability of species to respond to environmental changes. Tassel-eared squirrels are specialists of the ponderosa pine habitat, encountering naturally high levels of climate and resource variability across the year. Using collar-mounted accelerometer and GPS devices, we assessed the relative importance of weather and reproduction in determining levels of activity in a natural versus suburban (food supplemented) population of tassel-eared squirrels. In addition, we measured fecal metabolites of testosterone, estradiol, progesterone, and cortisol in squirrels to non-invasively assess patterns in reproductive timing, hormonal correlates of behavior, and glucocorticoid secretion between individuals and populations. While weather was a strong predictor of daily activity levels in both populations, seasonal variation in activity levels were more attenuated in squirrels from the suburban site when compared to the natural site. Furthermore, home range size remained relatively stable across the year at the suburban site but fluctuated seasonally at the natural site. Hormone analyses and field observations suggest that populations did not differ in reproductive timing or patterns of seasonal weight gain; however, we found higher cortisol and lower testosterone levels in the natural compared to suburban populations. Lastly, we found no relationships between testosterone/cortisol and indices of animal behavior. Taken together, the activity patterns of squirrels appear to be primarily driven by weather and seasonal fluctuations in food availability. Comparisons between sites suggest that anthropogenic food sources may drive endocrine and behavioral changes associated with a central place foraging strategy.