We determined the effect of fat adaptation on metabolism and performance during 5 h of cycling in seven competitive athletes who consumed a standard carbohydrate (CHO) diet for 1 day and then either a high-CHO diet (11 g · kg⁻¹ · day⁻¹ CHO, 1 g · kg⁻¹ · day⁻¹ fat; HCHO) or an isoenergetic high-fat diet (2.6 g · kg⁻¹ · day⁻¹ CHO, 4.6 g · kg⁻¹ · day⁻¹ fat; fat-adapt) for 6 days. On day 8, subjects consumed a high-CHO diet and rested. On day 9, subjects consumed a preexercise meal and then cycled for 4 h at 65% peak O₂ uptake, followed by a 1-h time trial (TT). Compared with baseline, 6 days of fat-adapt reduced respiratory exchange ratio (RER) with cycling at 65% peak O₂ uptake [0.78 ± 0.01 (SE) vs. 0.85 ± 0.02; P < 0.05]. However, RER was restored by 1 day of high-CHO diet, preexercise meal, and CHO ingestion (0.88 ± 0.01; P < 0.05). RER was higher after HCHO than fat-adapt (0.85 ± 0.01, 0.89 ± 0.01, and 0.93 ± 0.01 for days 2, 8, and 9, respectively; P < 0.05). Fat oxidation during the 4-h ride was greater (171 ± 32 vs. 119 ± 38 g; P < 0.05) and CHO oxidation lower (597 ± 41 vs. 719 ± 46 g; P < 0.05) after fat-adapt. Power output was 11% higher during the TT after fat-adapt than after HCHO (312 ± 15 vs. 279 ± 20 W; P = 0.11). In conclusion, compared with a high-CHO diet, fat oxidation during exercise increased after fat-adapt and remained elevated above baseline even after 1 day of a high-CHO diet and increased CHO availability. However, this study failed to detect a significant benefit of fat adaptation to performance of a 1-h TT undertaken after 4 h of cycling.