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Background: Voluntary wheel running (WVR) is a popular experimental tool to alter rodent activity levels and hypothetically increase activity energy expenditure (EE). Mice are typically housed at temperatures below the thermoneutral point, resulting in increased basal EE. However, whether VWR results in different metabolic phenotypes at different housing temperatures is unknown. Methods: Herein we assessed 7-day energy metabolism (indirect calorimetry), HFHS-induced weight gain, and body composition changes (qMRI) in male and female mice with running wheels or sedentary (SED) at either 20°C or 30°C. Results: LFD male and female mice housed at 30°C have ~40% lower total EE and 60% lower resting EE compared to 20°C mice. Only 30°C female SED mice had lower total and resting EE than males. VWR increased total EE regardless of temperature or sex. No difference was observed in activity EE by sex. 30°C mice had ~60% and ~40% greater activity EE compared to 20°C SED and VWR, respectively. Therefore, activity EE comprising ~28% and ~38% of total EE in 30°C SED and VWR, respectively, and ~10% and ~17% in 20°C SED and VWR, respectively. Male 30°C SED gained ~65% more weight during HFHS feeding, with VWR reducing weight gain ~40% in both groups. Interestingly, while HFHS-induced weight gain was ~35% greater in 30°C female SED and VWR mice compared to 20°C, VWR had no impact on weight gain in female mice of either temperature. These findings were mirrored in diet-induced change in fat mass. Importantly, no difference was observed in change in fat-free mass (FFM) in SED mice by temperature or sex. However, at 20°C & 30°C VWR resulted in reduced FFM gain in males and increased FFM gain in female mice. Conclusions: These data suggest that sex is a powerful mediator of the impact of increased physical activity, and thus EE, on metabolic outcomes. Further, these data suggest that thermoneutral housing provides a more appropriate experimental design to assess the role of activity EE on diet-induced disease phenotypes.