The purpose of this study was to assess oxygenation and neuromuscular activation of the quadriceps femoris muscle group, including the vastus intermedius (VI), during a fatiguing contraction. Eleven healthy men performed an isometric knee extension at 50% of the maximum voluntary contraction until failure. Near-infrared spectroscopy was used to measure tissue oxygen saturation (StO2 ), and surface electromyography was used to quantify the median frequency (MF) of the four individual quadriceps femoris muscles. A significant decrease in StO2 began at 25% of time to exhaustion in all four muscles, and StO2 of the VI was significantly higher than StO2 of the vastus medialis (VM) from 25% of time to exhaustion until 95% of time to exhaustion. The MF of the VI did not significantly decrease during the fatiguing task. We found a significant correlation between ∆MF and ∆StO2 in the VI (r=0·745, P<0·01) and a significant correlation between StO2 and time to exhaustion in the VM and rectus femoris (RF) (VM, r=0·684, P<0·05; RF, r=0·635, P<0·05). These results suggest that muscle O2 saturation in the VI is associated with neuromuscular fatigue during a fatiguing isometric contraction, and the level of oxygenation in the VM and RF is a key to sustaining a fatiguing contraction.