Development of functional near infrared spectroscopy (fNIRS) technologies has advanced quantification signal using multiple wavelength and detector to investigate hemodynamic response in human muscle. These non-invasive technologies have been widely used to solve the propagation of light inside the tissues including the absorption, scattering coefficient and to quantify the oxygenation level of haemoglobin and myoglobin in human muscle. The goal of this paper is to optimize the measurement of muscle oxygen saturation during isometric exercise using functional near infrared spectroscopy (fNIRS). The experiment was carried out on 15 sedentary healthy male volunteers. All volunteers are required to perform an isometric exercise at three assessment of muscular fatigue’s level on flexor digitalis (FDS) muscle in the human forearm using fNIRS. The slopes of the signals have been highlighted to evaluate the muscle oxygen saturation of regional muscle fatigue. As a result, oxygen saturation slope from 10% exercise showed steeper than the first assessment at 30%-50% of fatigues level. The hemodynamic signal response showed significant value (p=0.04) at all three assessment of muscular fatigue’s level which produce a p-value (p<0.05) measured by fNIRS. Thus, this highlighted parameter could be used to estimate fatigue’s level of human and could open other possibilities to study muscle performance diagnosis.