The aim of this study is to investigate the mechanism behind the slow increase in fat oxidation on a high-fat diet. Therefore, we determined 24-h substrate oxidation using respiration chambers and the rate of appearance and oxidation of plasma-derived fatty acids in seven healthy nonobese men (age 23 +/- 2 years, height 1.85 +/- 0.03 m, weight 70.4 +/- 2.3 kg, % body fat 13 +/- 1). Before testing, they consumed a low-fat diet (30% fat, 55% carbohydrate) at home for 3 days. Measurements were performed after 1 day consumption of either a low-fat diet (LF), a high-fat diet (HF1, 60% fat, 25% carbohydrate), or a high-fat diet preceded by a glycogen-lowering exercise test (HF1+EX), and after 7 days on a high-fat diet (HF7). After an overnight fast., an infusion of [U-C-13]palmitate (0.00806 mu mol . min(-1) . kg(-1)) was started and continued for 2 h at rest followed by 1 h of exercise at 50% of maximal power output (W-max). Whole-body fat oxidation was measured using indirect calorimetry and plasma-derived fatty acid oxidation was evaluated by measuring breath (CO2)-C-13 enrichment and corrected with the acetate recovery factor. Twenty-four-hour fat oxidation gradually increased on the high-fat diet. Both at rest and during exercise, there was no change in rate of appearance of fatty acids and plasma-derived fatty acid oxidation. Triglyceride-derived fatty acid oxidation tended to be higher after 7 days of high-fat diet at rest (P <0.07). This difference was significant during exercise (P <0.05). In conclusion, the results from this study suggest that triglyceride-derived fatty acid oxidation VLDL or intramuscular triglycerides) plays a role in the increase in fat oxidation on a high-fat diet, but plasma-derived fatty acids remain the major source for fat oxidation.