The aim of the present study was to examine the influence of training state on fasting-induced skeletal muscle pyruvate dehydrogenase (PDH) regulation, including PDH phosphorylation. Trained and untrained subjects, matched for skeletal muscle CS activity and OXPHOS protein, fasted for 36 h after receiving a standardized meal. Respiratory exchange ratio (RER) was measured and blood as well as vastus lateralis muscle biopsies were obtained 2, 12, 24, and 36 h after the meal. RER decreased with fasting only in untrained individuals, while PDHa activity decreased from 12 h after the meal in untrained, but only tended to decrease at 36 h in trained. PDH-E1α, PDP1 protein, PDH phosphorylation, and PDH acetylation in skeletal muscle was higher in trained than untrained subjects, but did not change with fasting, while PDK4 protein was higher at 36 h than at 2 h after the meal in both groups. In conclusion, the present results suggest that endurance exercise training modifies the fasting-induced regulation of PDHa activity in skeletal muscle and the substrate switch towards fat oxidation. PDH phosphorylation could not explain the fasting-induced regulation of PDHa activity suggesting other post translational modifications.