The effect of endurance training on 24-h energy expenditure (EE), basal metabolic rate (BMR), sleeping metabolic rate (SMR), and the thermic effect of food (TEF) was assessed in a respiratory chamber where only spontaneous physical activity (SPA) was allowed. Results from 20 highly trained male endurance athletes (25 +/- 5 yr, 178 +/- 7 cm, 70 +/- 8 kg body wt, 64 +/- 7 kg fat-free mass) were compared with those of 43 untrained males who were matched for age (28 +/- 6 yr), height (175 +/- 5 cm), weight (73 +/- 13 kg), and fat-free mass (62 +/- 8 kg). Subjects were admitted to a metabolic ward, fed a weight-maintenance diet, and refrained from physical activity for at least 2 days before measurements. No significant differences were found with respect to 24-h EE (2,126 +/- 186 vs. 2,154 +/- 245 kcal), BMR (1,808 +/- 342 vs. 1,709 +/- 329 kcal), SMR (1,523 +/- 120 vs. 1,555 +/- 188 kcal), or TEF (24.9 +/- 9.2 vs. 21.3 +/- 6.7% of ingested calories; these values included the energy cost of arousal) between trained and untrained subjects, respectively, before or after adjusting for differences in body composition. Neither the 24-h respiratory quotient nor the level of SPA differed between the two groups. No relationship was found between maximal aerobic capacity and metabolic rate adjusted for differences in fat-free mass and fat mass. These results do not support an effect of fitness level on EE measured under sedentary conditions. Therefore, although physical activity plays an important role in maintaining optimal health and body weight, its effect on calorie output does not extend beyond the cost of the activity and recovery periods.