The purpose of this study was to elucidate the fat-loading effect of the classic 1-wk glycogen-loading regimen histologically in rats and physiologically in humans. In the rat and human studies, an exhaustive swimming exercise and cycle ergometer exercise were loaded on day 1 of a 6-d feeding period, respectively. Thereafter, both the rats and humans were divided into a glycogen-loading regimen consisting of a 3-d high-fat diet and a 3-d high-carbohydrate diet or a 6-d high-carbohydrate diet. After the feeding period in the human study, the human subjects performed a test exercise on day 7 using a cycle ergometer. In the rat study, the intramuscular triglyceride (IMTG) content was 69% greater (p<0.05) after the glycogen-loading regimen than after the high-carbohydrate diet feeding on day 7. In the human study, the respiratory exchange ratios (RER) after the glycogen-loading regimen were 4.9-6% lower than those after the high-carbohydrate diet during the test exercise on day 7 (p<0.05). Our findings suggest that the classical 1-wk glycogen-loading regimen maintained the storage and enhanced the utilization of energy sources during exercise in the skeletal muscle, and that it provides a fat-loading effect, in addition to the glycogen-loading effect, to the skeletal muscle.