In order to obtain evidence that Mb releases O2 during muscle contraction, we have set up a buffer-perfused hindlimb rat model and applied NIRS to detect the dynamics of tissue deoxygenation during contraction. The NIRS signal was monitored on hindlimb muscle during twitch contractions at 1 Hz, evoked via electrostimulator at different submaximal levels. The hindlimb perfusion was carried out by perfusion of Krebs Bicarbonate buffer. The NIRS still detected a strong signal even under Hb-free contractions. The deoxygenation signal (D[deoxy]) was progressively increased at onset of the contraction and reached the plateau under both blood- and buffer-perfused conditions. However, the amplitude of D[deoxy] during steady state continued to significantly increase as tension increased. The tension-matched comparison of the D[deoxy] level under buffer-perfused and blood perfused conditions indicate that Mb can contribute approximately 50% to the NIRS signal. These results clarify the Mb contribution to the NIRS signal and show a falling intracellular PO2 as workload increases.