Objective: Test the hypothesis that FFA and muscle glycogen modify exercise-induced regulation of PDH in human skeletal muscle through regulation of PDK4 expression. Research Design and Methods: On two occasions, healthy male subjects lowered (by exercise) muscle glycogen in one leg (LOW) relative to the contra-lateral leg (CON) the day before the experiment day. On the experimental days, plasma FFA was ensured normal or remained elevated by consuming breakfast rich (low FFA) or poor (high FFA) in carbohydrate, 2 hours before performing 20 min of two-legged knee extensor exercise. Vastus lateralis biopsies were obtained before and after exercise. Results: PDK4 protein content was approximately 2.2 and approximately 1.5 fold higher in LOW than CON leg in high FFA and low FFA, respectively, and the PDK4 protein content in CON leg was approximately 2 fold higher in high FFA than in low FFA. In all conditions, exercise increased PDHa activity resulting in similar levels in LOW leg in both trials and CON leg in high FFA, but higher level in CON leg in low FFA. PDHa activity was closely associated with PDH-E1alpha phosphorylation. Conclusions: Muscle glycogen and plasma FFA attenuate exercise-induced PDH regulation in human skeletal muscle in a non-additive manner. This might be through regulation of PDK4 expression. The activation of PDH by exercise independent of changes in muscle glycogen or plasma FFA suggests that exercise overrules FFA-mediated inhibition of PDH (i.e. carbohydrate oxidation), and this may thus be one mechanism behind health promoting effects of exercise.