Multispectral imaging (MSI) is a new, non-invasive method to continuously measure oxygenation and microcirculatory perfusion, but has limitedly been validated in healthy volunteers. The present study aimed to validate the potential of multispectral imaging in the detection of microcirculatory perfusion disturbances during a vascular occlusion test (VOT). Two consecutive VOT’s were performed on healthy volunteers and tissue oxygenation was measured with MSI and near-infrared spectroscopy (NIRS). Correlations between the rate of desaturation, recovery and the hyperemic area under the curve (AUC) measured by MSI and NIRS were calculated. Fifty-eight volunteers were included. The MSI oxygenation curves showed identifiable components of the VOT, including a desaturation and recovery slope and hyperemic area under the curve, similar to those measured with NIRS. The correlation between the rate of desaturation measured by MSI and NIRS was moderate: r = 0.42 (p = 0.001) for the first and r = 0.41 (p = 0.002) for the second test. Our results suggest that non-contact multispectral imaging is able to measure changes in regional oxygenation and deoxygenation during a vascular occlusion test in healthy volunteers. When compared to measurements with NIRS, correlation of results was moderate to weak, most likely reflecting differences in physiology of the regions of interest and measurement technique.