This study compared the fat metabolism between "a single bout of prolonged exercise" and "repeated bouts of exercise" of equivalent exercise intensity and total exercise duration. Seven men performed three trials: 1) a single bout of 60 min exercise (Single); 2) two bouts of 30-min exercise, separated by a 20-min rest between exercise bouts (Repeated); and 3) rest. Each exercise was performed with a cycle ergometer at 60% of maximal oxygen uptake. In the Single and Repeated trials, serum glycerol, growth hormone, plasma epinephrine and norepinephrine concentrations increased significantly (P < 0.05) during the first 30-min exercise bout. In the Repeated trial, serum free fatty acids (FFA), acetoacetate and 3-hydroxybutyrate concentrations showed rapid increases (P < 0.05) during a subsequent 20-min rest period. During the second 30-min exercise bout, FFA and epinephrine responses were significantly greater in the Repeated trial than in the Single trial (P < 0.05). Moreover, the Repeated trial showed significantly lower values of insulin and glucose than in the Single trial. During the 60-min recovery period after the exercise, FFA, glycerol, and 3-hydroxybutyrate concentrations were significantly higher in the Repeated trial than in the Single trial (P < 0.05). The relative contribution of fat oxidation to the energy expenditure showed significantly higher values (P < 0.05) in the Repeated trial than in the Single trial during the recovery period. These results indicate that repeated bouts of exercise cause enhanced fat metabolism compared with a single bout of prolonged exercise of equivalent total exercise duration.