Pyruvate dehydrogenase (PDH) and lactate dehydrogenase (LDH) are essential for ATP production in skeletal muscle. However, directly PDH flux in exercising human muscle has been challenging and never assessed. This study was to demonstrate the feasibility of assessing PDH activation and changes in pyruvate metabolism in human skeletal muscle after the onset of exercise using hyperpolarized [1-13C]pyruvate. During moderate flexion-extension exercise, total HP 13C signals (tC), [1-13C]lactate/tC, and [13C]bicarbonate/tC increased significantly compared to resting state. This study demonstrates that PDH flux in skeletal muscle increases rapidly after the onset of exercise and decreases during recovery.