Objective: This study investigated the contributions of physical fitness and body composition to 24-h fat oxidation in adults under sedentary conditions in a whole-room calorimeter. Methods: The following measurements were studied in 109 adults (49 male/45 female) at least 36 h after a bout of exercise: 1) aerobic fitness level assessed by V̇O(2max), 2) body composition determined by underwater weighing, 3) resting metabolic rate (RMR) after an overnight fast, and 4) 24-h energy expenditure (EE) and substrate oxidation determined in a whole-room calorimeter. While in the calorimeter, subjects were provided with a diet (15% protein, 30% fat, and 55% carbohydrate) estimated to produce energy balance on a sedentary day and of similar nutritional composition to their daily dietary intake. Results: We found strong negative correlations between V̇O(2max) and % body fat in both male and female subjects, but no relationship between V̇O(2max) and 24-h EE under the sedentary conditions of this study. In male subjects, V̇O(2max) (mL O2·kg-1 fat-free mass·min-1) was negatively related to fat oxidation (r = -0.397, P < 0.005), and fat oxidation was more closely related to fat mass (r = 0.434, P < 0.0002) than to fat-free mass (r = 0.165, NS). In contrast, none of these relationships were significant in females. Conclusion: The results show that in male subjects under sedentary conditions, 24-h fat oxidation is positively related to body fat mass and negatively related to V̇O(2max) (the marker used here for level of physical fitness). This supports our hypothesis that regularly active males maintain lower body fat stores as the low contribution to daily fat oxidation from a lower body fat mass is counterbalanced by the high contribution to fat oxidation from daily physical activity. The lack of a relationship between V̇O(2max) and 24-h EE under the sedentary conditions of this study suggests that the major effects of physical activity on total daily EE and fat oxidation may occur during and relatively quickly after an exercise bout. Further, these data also suggest that cessation of regular exercise will likely be associated with a high risk of positive fat balance and weight gain.