This study aimed to compare changes in skeletal muscle, prefrontal (PFC), and motor (MC) cortex hemodynamics during prolonged (i.e., 4-h) fatiguing whole-body exercise using multichannel near-infrared spectroscopy (NIRS). Ten subjects completed three successive 80-min cycling bouts at 45 % of their maximal power output. After the 4-h cycling, maximal voluntary contraction force of the leg was decreased by ~25 %. Muscle exhibited reproductive deoxygenation patterns during each of the three bouts, whereas intra-bout cerebral hemodynamics were different throughout the protocol. Results demonstrate that specific responses to fatiguing exercise are found between tissues but also between cortical sites involved in cycling, as shown by concomitant PFC hyperoxygenation and MC deoxygenation in the first 80 min of exercise. Further insights are needed to understand the consequences of these changes regarding the integrative control of motor output while fatigue develops over several hours.