Five days of a high fat diet while training, followed by 1 day of carbohydrate (CHO) restoration, increases rates of whole-body fat oxidation and decreases CHO oxidation during aerobic cycling. The mechanisms responsible for these shifts in fuel oxidation are unknown, but would involve up and down regulation of key regulatory enzymes in the pathways of skeletal muscle fat and CHO metabolism, respectively. This study measured muscle pyruvate dehydrogenase (PDH) and hormone sensitive lipase (HSL) activities pre- and post 20 min of cycling at 70% of VO_2peak and 1 min of sprinting at 150% peak power output (PPO). Estimations of muscle glycogenolysis were made during the initial min of exercise at 70% VO_2peak and during the 1 min sprint. Seven male cyclists undertook this exercise protocol on two occasions. For 5 days, subjects consumed in random order either a high-CHO (HCHO) diet (10.3 g^.kg^-1.day^-1 CHO or ~70% of total energy intake) or an isoenergetic high-fat (FAT-adapt) diet (4.6 g^.kg^-1.day^-1 FAT or 67% of total energy) while undertaking supervised aerobic endurance training. On day 6 for both treatments, subjects ingested a high CHO diet and rested before their experimental trials on day 7. This CHO restoration resulted in similar resting glycogen contents (FAT-adapt: 873 ± 121 vs. HCHO: 868 ± 120 umoles glucosyl units ^.g^-1 dw). However, the respiratory exchange ratio was lower during cycling at 70% VO₂ peak in the FAT-adapt trial, which resulted in a ~45% increase and a ~30% decrease in fat and CHO oxidation respectively. PDH activity was lower at rest and throughout exercise at 70% VO_2peak (1.69 ± 0.25 vs 2.39 ± 0.19 mmol^.kg^-1 ww^.min^-1) and the 1 min sprint in the FAT-adapt compared to HCHO trial. Estimates of glycogenolysis during the first min of exercise at 70% VO_2peak and the 1 min sprint were also lower following FAT-adapt (9.1 ± 1.1 vs 13.4 ± 2.1 and 37.3 ± 5.1 vs 50.5 ± 2.7 glucosyl units^.kg^-1 dw^.min^-1). HSL activity was ~20% higher (P=0.12) during exercise at 70% VO_2peak following FAT-adapt. These results indicate that the previously reported decreases in whole body CHO oxidation and increases in fat oxidation following the FAT-adapt protocol are a function of metabolic changes within skeletal muscle. The metabolic signals responsible for the shift in muscle substrate use during cycling at 70% VO_2peak remain unclear but lower accumulation of free ADP and AMP following the FAT-adapt trial may be responsible for the decreased glycogenolysis and PDH activation during sprinting.