Near-infrared spectroscopy (NIRS) uses the relative absorption of light at 850 and 760 nm to determine skeletal muscle oxygen saturation. Previous studies have used the ratio of both signals to report muscle oxygen saturation. Purpose: The purpose of this pilot study is to assess the different approaches used to represent muscle oxygen saturation and to evaluate the pulsations of oxygenated hemoglobin/myoglobin (O2
heme) and deoxygenated hemoglobin/myoglobin (Heme) signals. Method: Twelve participants, aged 20–29 years, were tested on the forearm flexor muscles using continuous-wave NIRS at rest. Measurements were taken during 2–3 min rest, physiological calibration (5 min ischemia), and reperfusion. Ten participants were included in the study analysis. Results: There was a significant difference in pulse size between O2
heme and Heme signals at the three locations (p
< 0.05). Resting oxygen saturation was 58.8% + 9.2%, 69.6% + 3.9%, and 89.2% + 6.9% when calibrated using O2
heme, the tissue oxygenation/saturation index (TSI), and Heme, respectively. Conclusion: The difference in magnitude of O2
heme and Heme pulses with each heartbeat might suggest different anatomical locations of these signals, for which calibrating with just one of the signals instead of the ratio of both is proposed. Calculations of physiological calibration must account for increased blood volume in the tissue because of the changes in blood volume, which appear to be primarily from the O2
heme signal. Resting oxygen levels calibrated with Heme agree with theoretical oxygen saturation.