The present study investigated whether or not muscle triacylglycerol (MTG) contributed as a main energy source and MTG level and utilized fatty acid (FA) composition decreased during a 4-hour swimming exercise in rats fed a normal diet or a high-fat diet (HFD). Sixty male Wistar rats aged 5 weeks were fed a normal diet (CE-2, n = 25, experiment A) or HFD (n = 35, experiment B) for 22 days. On the final day, rats in both experiments were killed either without exercise or 1, 2, 3, or 4 hours after beginning the swimming exercise. MTG accumulation was higher in rats fed the HFD than those fed the CE-2 in both slow-and fast-typed muscles. Serum concentrations of free fatty acids (FFA) and glucose were increased and muscle glycogen contents were decreased with the continuance of swimming exercise, especially in rats fed the CE-2. The prolonged swimming did not influence MTG contents and FA compositions of MTG in either the experiment. These results might indicate that specific FA of MTG was not oxidized and MTG did not contribute as a main energy source during the prolonged swimming exercise in rats; instead, serum FFA, glucose, and muscle glycogen were mainly used.