We investigated the test–retest reliability of, and age-related differences in, markers of skeletal muscle metabolism and microvascular function derived from the near-infrared spectroscopy with vascular occlusion test (NIRS-VOT) in younger women (YW) and middle-aged and older women (MAOW). Seventeen YW (age 23 ± 4 years) and 17 MAOW (age 59 ± 8 years) completed this study. Participants completed identical experimental visits separated by ∼4 weeks during which the NIRS-VOT was used to quantify the occlusion slope, minimum and maximum tissue saturation, ischaemic index, reperfusion magnitude, the reperfusion and 10-s reperfusion slopes (slope 2 and slope 210-s), time to max tissue saturation, and area under the reperfusion curve using the local tissue oxygen saturation signal. Except for slope 210-s (intraclass correlation coefficient (ICC) = 0.37; coefficient of variation (CV) = 31%), time to max tissue saturation (ICC = 0.21), and ischaemic index (ICC = 0.37) for MAOW, all of the NIRS variables demonstrated good to excellent relative reliability for the YW (ICCs = 0.74–0.86) and the MAOW (ICCs = 0.51–0.87), with CVs of 2–21% and 2–22%, respectively. The occlusion slope was significantly lower, indicating accelerated deoxygenation, while maximum tissue saturation, reperfusion magnitude, and ischaemic index were significantly higher in YW versus MAOW. No other group differences were found. In conclusion, our data support the use of the NIRS-VOT as a simple, reliable, non-invasive technique for the assessment of peripheral skeletal muscle metabolism and microvascular function in women, with the reliability being generally greater in YW versus MAOW. Further, our data suggest that ageing is associated with lower skeletal muscle metabolism and microvascular hyperaemic responsiveness in women.