Background: Free fatty acids (FFAs) are an important source of energy, and also serve as signaling molecules to regulate gene expression. Exercise performed in a post-absorptive state, in contrast to that performed in a postprandial state, increases 24-h fat oxidation under an energy-balanced condition. The primary aim of the present study was to clarify whether the effects of exercise on the concentration and composition of plasma FFAs, which may underlie distinct effects of exercise on 24-h fat oxidation, depend on the nutritional state of the individual when performing the exercise. Methods: Ten healthy young men underwent 3 trials of indirect calorimetry in a metabolic chamber. The subjects performed exercise at 60% of VO2max for 60 min in either a post-absorptive or postprandial state, or remained sedentary without an exercise session (control). All trials were designed to be energy balanced over 24 h. Blood samples were collected immediately before and after exercise. Results: Fat oxidation over 24 h was increased only when exercise was performed in a post-absorptive state (control, 531 ± 60; post-absorptive, 779 ± 70; postprandial, 569 ± 37 kcal/24 h). The increase in the 24-h fat oxidation was related to the magnitude of the transient carbohydrate deficit after exercise. The plasma FFA concentration after exercise was higher in the post-absorptive trial (0.38 ± 0.04) than in the control (0.13 ± 0.01) and postprandial (0.15 ± 0.02 mM) trials. The ratio of unsaturated to saturated (U/S) fatty acids after exercise was higher in the post-absorptive trial (1.76 ± 0.06) than in the control (1.56 ± 0.07) and postprandial (1.53 ± 0.08) trials. On the other hand, the plasma FFA concentration after exercise in a postprandial state did not differ significantly from that in the control trial. Conclusion: Exercise performed in a post-absorptive state effectively increased the plasma FFA concentration and U/S ratio to a greater degree than exercise performed in a postprandial state, underlying the increase in the 24-h fat oxidation. The increase in the plasma FFA concentration was related to the transient carbohydrate deficit after exercise.