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 = 10, body mass index (BMI) = 22.5 ± 6.5 kg/m2], lean endurance-trained (LT, n = 10, BMI = 21.2 ± 1.2 kg/m2), and obese sedentary (OS, n = 7, BMI = 35.5 ± 4.4 kg/m2) 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 ± 9 vs. 29 ± 7 g/day, respectively), LT (53 ± 8 vs. 42 ± 5 g/day), or OS (58 ± 7 vs. 80 ± 9 g/day). However, 24-h fat balance was significantly more positive on EX compared with CON (P < 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. Copyright © 2009 the American Physiological Society.