Acute high-fat (HF) diets can lead to short-term positive fat balances until the body increases fat oxidation to match intake. The purpose of this study was to examine the effects of a HF diet, rich in either mono-unsaturated or saturated fatty acids (FAs) and exercise, on the rate at which the body adapts to a HF diet.(13)C-labeled oleate and (2)H-labeled palmitate were also given to determine the contribution of exogenous vs. global fat oxidation. Eight healthy men (age of 18-45 yr; body mass index of 22 ± 3 kg/m(2)) were randomized in a 2 × 2 crossover design. The four treatments were a high saturated fat diet with exercise (SE) or sedentary (SS) conditions and a high monounsaturated fat diet with exercise (UE) or sedentary (US) conditions. Subjects stayed for 5 days in a metabolic chamber. All meals were provided. On day 1, 30% of energy intake was from fat, whereas days 2-5 had 50% of energy as fat. Subjects exercised on a stationary cycle at 45% of maximal oxygen uptake for 2 h each day. Respiratory gases and urinary nitrogen were collected to calculate fat oxidation. Change from day 1 to day 5 showed both exercise treatments increased fat oxidation (SE: 76 ± 30 g, P = 0.001; UE: 118 ± 31 g, P < 0.001), whereas neither sedentary condition changed fat oxidation (SS: -10 ± 33 g, P = not significant; US: 41 ± 14 g, P = 0.07). No differences for dietary FA composition were found. Exercise led to a faster adaptation to a HF diet by increasing fat oxidation and achieving fat balance by day 5. Dietary FA composition did not differentially affect 24-h fat oxidation.