Exercise-induced energy expenditure (ExEE) is commonly adopted as a mean to volitional body mass loss. ExEE can also modulate physical activity energy expenditure (PAEE) and potentially limit the rate of body mass loss. The detrimental effect of ExEE on PAEE may be more prevalent in women than in men. This study was to investigate the sex differences in regulation of PAEE and body mass change following an acute exercise-induced perturbation in energy balance.  In this 3-week study, 4 men and 6 women with body mass index (BMI) of 28.8 ± 2.3 kg/m² were required to exercise on a leg ergometer, expending 15% of their total daily energy expenditure (TDEE) in each exercise session of week two (Exercise week). Participants had to complete three exercise sessions within the same week and on non-consecutive days. PAEE was assessed via wrist-worn accelerometers over the entire study period. Resting metabolic rate (RMR) and body composition were measured at the beginning of the study. Men had significantly greater stature (p=0.021), body mass (p=0.035) and resting metabolic rate (RMR) (p=0.01) than women. Whereas women body fat % was significantly greater than men (p=0.004). At the end of the study, there was no significant difference body mass % change (p=0.409) and ExEE (p=0.91) between men and women. There was no significant main effect of time for PAEE (p=0.613), or between sex (p=0.470). Three bouts of ExEE performed within the same week did not alter PAEE in men and women with overweight and obesity.