Near-infrared spectroscopy (NIRS) is a noninvasive way of measuring muscular oxygenation. We evaluated the relationship between NIRS signal [infrared muscle oxygen saturation (IR-SO2mus)] and the femoral venous oxygen saturation (SfvO2) during cycling exercise. Six healthy subjects performed a 30-min steady-state exercise at 80% maximal oxygen uptake in normoxia and hypoxia (inspired O2 fraction = 0.105). IR-So2mus was recorded continuously throughout the tests with the NIRS probe located on the vastus lateralis. During exercise, blood samples were withdrawn every 5 min from radial artery and femoral vein catheters. In normoxia, IR-So2mus initiated a transient nonsignificant decrease at 5 min, then returned to preexercise level, whereas SfvO2 showed a fast decrease, reaching 18% saturation at 10 min without further change. By contrast, in hypoxia, IR-SO2mus and SfvO2 demonstrated a parallel decrease then stabilized at 10 min. We conclude that IR-SO2mus appears to parallel SfvO2 when both the arterial and venous oxygen contents decrease during steady-state exercise in hypoxia, whereas IR-SO2mus does not follow SfvO2 change in normoxia.