This study investigated the influence of different durations of aerobic exercise on fuel utilization, lactate levels and antioxidative status in trained rats. Forty rats underwent physical training (T, n = 20) or non- training (NT, n = 20) for 6 weeks. For physical training, animals exercised on a treadmill for 30 min 5 days per week. At the end of week 6, the animals in each group were subdivided into BE, DE-0.5, DE-1 and DE-2, which were sacrificed at the end of week 6 without having performed exercise or after exercise on a treadmill for 0.5h, 1h and 2h, respectively, immediately before being sacrificed. The plasma glucose level in DE-2 of the NT group was significantly lower than in the other groups. Muscle and liver glycogen levels were significantly lower in DE-1 and DE-2, but there were no significant differences between DE-1 and DE-2 in the T group. Liver protein in DE-2 of the NT group was significantly lower. Muscle TG levels were decreased in DE-0.5 of the T group, while those of the NT group were decreased in DE-1. FFA levels were increased in DE-0.5 of the T group and in DE-1 of the NT group. Lactate levels were increased in DE-0.5 of the NT group, while they were increased in DE-1 of the T group. Catalase activity of the T group was lower in BE but higher in DE-0.5, DE-1 and DE-2. SOD activities were higher in trained rats, while the GSH/GSSG ratios were higher in BE, DE-0.5 and DE-1 in the T group, and there was no difference in that of DE-2. There were no differences in MDA levels in BE and DE-0.5, but they were significantly lower in DE-1 and DE-2 of the T group. Overall, the results of this study, suggest that training may improve exercise performance by facilitating the mobilization and oxidation of fat and conserving limited carbohydrate storage, and that it may delay the onset of fatigue and enhance the antioxidative defense system, but cannot support two hours of vigorous exercise.