Significance: Hemoglobin oxygen saturation and red blood cell (RBC) tissue fraction are important parameters when assessing microvascular status. Functional information can be attained using temporally resolved measurements performed during stimulus–response protocols. Pointwise assessments can currently be conducted with probe-based systems. However, snapshot multispectral imaging (MSI) can be used for spatial–temporal measurements.

Aim: To validate if hemoglobin oxygen saturation and RBC tissue fraction can be quantified using a snapshot MSI system and an inverse Monte Carlo algorithm.

Approach: Skin tissue measurements from the MSI system were compared to those from a validated probe-based system during arterial and venous occlusion provocation on 24 subjects in the wavelength interval 450 to 650 nm, to evaluate a wide range of hemoglobin oxygen saturation and RBC tissue fraction levels.

Results: Arterial occlusion results show a mean linear regression R²  =  0.958 for hemoglobin oxygen saturation. Comparing relative RBC tissue fraction during venous occlusion results in R²  =  0.925. The MSI system shows larger dynamic changes than the reference system, which might be explained by a deeper sampling including more capacitance vessels.

Conclusions: The snapshot MSI system estimates hemoglobin oxygen saturation and RBC tissue fraction in skin microcirculation showing a high correlation (R²  >  0.9 in most subjects) with those measured by the reference method.