Abstract Objective Fat oxidation during exercise depends on nutritional state, and exercise performed in the post-absorptive state oxidizes more fat than that performed in the postprandial state. However, the effects of exercise on energy metabolism continue during the post-exercise period, and the difference in fat oxidation during exercise may be compensated for during the post-exercise period. The present study compared the effects of an acute exercise bout in the post-absorptive or postprandial state on 24h fat oxidation. Methods Twelve young male athletes stayed twice in a room-size metabolic chamber for 24h indirect calorimetry in a randomized repeated-measure design. Before or after breakfast, i.e. in the post-absorptive or postprandial state, subjects exercised at 50% VO2 max for 60min. Results During the 60min of exercise, energy expenditure in the two exercise trials were equivalent, but exercise in the post-absorptive state was performed with lower RQ compared with that in the postprandial state (P<0.01). The time of exercise relative to breakfast did not affect 24h energy expenditure (P>0.5). However, accumulated 24h fat oxidation was higher (P<0.05) and that of carbohydrate oxidation was lower (P<0.05) when exercise was performed in the post-absorptive state. Conclusions Compared with exercise performed in the postprandial state, exercise performed in the post-absorptive state oxidized more fat and saved more carbohydrate in the body, without affecting 24h energy expenditure.