The purpose of this investigation was to examine the effects of diet on muscle triglyceride and endurance performance. Seven endurance-trained men completed a 120-min cycling bout at 65% of maximal oxygen uptake. Each subject then ingested an isocaloric high-carbohydrate (Hi-CHO; 83% of energy) or a high-fat (Hi-Fat; 68% of energy) diet for the ensuing 12 h. After a 12-h overnight fast, a 1,600-kJ self-paced cycling bout was completed. Muscle triglyceride measured before (33.0 +/- 2.3 vs. 37.0 +/- 2.1 mmol/kg dry wt) and after (30.9 +/- 2.4 vs. 32.8 +/- 1.6 mmol/kg dry wt) the 120-min cycling bout was not different between the Hi-CHO and Hi-Fat trials, respectively. After the 24-h dietary-fasting period, muscle triglyceride was significantly higher for the Hi-Fat (44.7 +/- 2.4 mmol/kg dry wt) vs. the Hi-CHO (27.5 +/- 2.1 mmol/kg dry wt) trial. Furthermore, self-paced cycling time was significantly greater for the Hi-Fat (139.3 +/- 7.1 min) compared with the Hi-CHO (117.1 +/- 3.2 min) trial. These data demonstrate that there was not a significant difference in muscle triglyceride concentration before and after a prolonged moderate-intensity cycling bout. Nevertheless, a high-fat diet increased muscle triglyceride concentration and reduced self-paced cycling performance 24 h after the exercise compared with a high-carbohydrate diet.