Activity energy expenditure (AEE) is the component of daily energy expenditure that is mainly influenced by the amount of physical activity (PA) and by the weight of the body displaced. This study aimed at analyzing the effect of weight loss on PA and AEE. The body weight and PA of 66 overweight and obese subjects were measured at baseline and after 12 weeks of 67% energy restriction. PA was measured using a tri-axial accelerometer for movement registration (Tracmor) and quantified in activity counts. Tracmor recordings were also processed using a classification algorithm to recognize 6 common activity types engaged in during the day. A doubly-labeled water validated equation based on Tracmor output was used to estimate AEE. After weight loss, body weight decreased by 13±4%, daily activity counts augmented by 9% (95% CI: +2%, +15%), and this increase was weakly associated with the decrease in body weight (R2 = 7%; P<0.05). After weight loss subjects were significantly (P<0.05) less sedentary (-26 min/d), and increased the time spent walking (+11 min/d) and bicycling (+4 min/d). However, AEE decreased by 0.6±0.4 MJ/d after weight loss. On average, a 2-hour/day reduction of sedentary time by increasing ambulatory and generic activities was required to restore baseline levels of AEE. In conclusion, after weight loss PA increased but the related metabolic demand did not offset the reduction in AEE due to the lower body weight. Promoting physical activity according to the extent of weight loss might increase successfulness of weight maintenance.