We hypothesized that near-infrared spectroscopy (NIRS) measures of hemoglobin and/or myoglobin O2 saturation (IR-So 2) in the vascular bed of exercising muscle would parallel changes in femoral venous O2 saturation (S[Formula: see text]) at the onset of leg-kicking exercise in humans. Six healthy subjects performed transitions from rest to 48 ± 3 (SE)-W two-legged kicking exercise while breathing 14, 21, or 70% inspired O2. IR-So 2 was measured over the vastus lateralis muscle continuously during all tests, and femoral venous and radial artery blood samples were drawn simultaneously during rest and during 5 min of exercise. In all gas-breathing conditions, there was a rapid decrease in both IR-So 2 and SfvO2 at the onset of moderate-intensity leg-kicking exercise. Although SfvO2 remained at low levels throughout exercise, IR-So 2increased significantly after the first minute of exercise in both normoxia and hyperoxia. Contrary to the hypothesis, these data show that NIRS does not provide a reliable estimate of hemoglobin and/or O2 saturation as reflected by direct femoral vein sampling.