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Background There is a spectrum of possibilities for analyzing muscle O2 resaturation parameters for measurement of reactive hyperemia in microvasculature. However, there is no consensus with respect to the responsiveness of these O2 resaturation parameters for assessing reactive hyperemia. Objectives This study investigates the responsiveness of the most utilized muscle O2 resaturation parameters to assess reactive hyperemia in the microvasculature of a clinical group known to exhibit impairments of tissue O2 saturation (StO2). Methods Twenty-three healthy young adults, twenty-nine healthy older adults, and thirty-five older adults at risk of cardiovascular disease (CVD) were recruited. Near-infrared spectroscopy (NIRS) was used to assess StO2 after a 5-min arterial occlusion challenge and the following parameters were analyzed: StO2slope_10s, StO2slope_30s, and StO2slope_until_baseline (upslope of StO2 over 10s and 30s and until StO2 reaches the baseline value); time to StO2baseline and time to StO2max (time taken for StO2 to reach baseline and peak values, respectively); ∆StO2reperfusion (the difference between minimum and maximum StO2 values); total area under the curve (StO2AUCt); and AUC above the baseline value (StO2AUC_above_base). Results Only StO2slope_10s was significantly slower in older adults at risk for CVD compared to healthy young individuals (p < 0.001) and to healthy older adults (p < 0.001). Conversely, time to StO2max was significantly longer in healthy young individuals than in older adult at CVD risk. Conclusions Our findings suggest that StO2slope_10s may be a measure of reactive hyperemia, which provides clinical insight into microvascular function assessment.