. When energy balance is maintained, exercise does not induce negative fat balance in lean sedentary, obese sedentary, or lean endurance-trained individuals. J Appl Physiol 107: 1847-1856, 2009. First published October 15, 2009 doi:10.1152/japplphysiol.00958.2009.-Fat oxidation during exercise is increased by endurance training, and evidence suggests that fat oxidation during exercise is impaired in obesity. Thus the primary aim of this study was to compare the acute effects of exercise on 24-h fat oxidation and fat balance in lean sedentary [LS, n ϭ 10, body mass index (BMI) ϭ 22.5 Ϯ 6.5 kg/m 2 ], lean endurance-trained (LT, n ϭ 10, BMI ϭ 21.2 Ϯ 1.2 kg/m 2 ), and obese sedentary (OS, n ϭ 7, BMI ϭ 35.5 Ϯ 4.4 kg/m 2 ) men and women. Twenty-four-hour energy expenditure and substrate oxidation were measured under sedentary (control; CON) and exercise (EX) conditions while maintaining energy balance. During EX, subjects performed 1 h of stationary cycling at 55% of aerobic capacity. Twenty-four-hour fat oxidation did not differ on the CON or EX day in LS (43 Ϯ 9 vs. 29 Ϯ 7 g/day, respectively), LT (53 Ϯ 8 vs. 42 Ϯ 5 g/day), or OS (58 Ϯ 7 vs. 80 Ϯ 9 g/day). However, 24-h fat balance was significantly more positive on EX compared with CON (P Ͻ 0.01). Twenty-four-hour glucose, insulin, and free fatty acid (FFA) profiles were similar on the EX and CON days, but after consumption of the first meal, FFA concentrations remained below fasting levels for the remainder of the day. These data suggest that when exercise is performed with energy replacement (i.e., energy balance is maintained), 24-h fat oxidation does not increase and in fact, may be slightly decreased. It appears that the state of energy balance is an underappreciated factor determining the impact of exercise on fat oxidation. whole room calorimeter; fat oxidation; body weight regulation IMPAIRMENTS in fat metabolism contribute to the development of obesity and non-insulin-dependent diabetes mellitus. For example, reduced rates of 24-h fat oxidation [i.e., increased respiratory quotient (RQ)] have been shown to predict weight gain (39), and obese and insulin-resistant individuals have a decreased capacity to oxidize fatty acids (20). Moreover, fatty acid oxidation in isolated skeletal muscle preparations from obese individuals is lower than in muscle from lean individuals (23). Fat oxidation is increased during exercise, and endurance training promotes changes in skeletal muscle that would favor an increase in fat oxidation (19), suggesting that regular endurance exercise could induce loss of fat mass by increasing fat oxidation. It has been suggested that exercise substantially increases fat oxidation in the postexercise period (13), although this has never clearly been demonstrated.To reduce fat mass, a state of negative fat balance must be achieved (11). To achieve negative fat balance, one must alter intake or expenditure such that fat oxidation exceeds fat intake. Since fat oxidation is increased during exercise, it is generally assumed that more fat is o...