Low muscle oxidative capacity contributes to exercise intolerance in chronic obstructive pulmonary disease (COPD). Near-infrared spectroscopy (NIRS) allows non-invasive determination of the muscle oxygen consumption (mV̇O2) recovery rate constant (k), which is proportional to oxidative capacity assuming two conditions are met: 1) exercise intensity is sufficient to fully-activate mitochondrial oxidative enzymes; 2) sufficient O2 availability. We aimed to determine reproducibility (coefficient of variation, CV; intraclass correlation coefficient, ICC) of NIRS k assessment in the gastrocnemius of 64 participants with (FEV1 64 ± 23%predicted) or without COPD (FEV1 98 ± 14%predicted). 10–15 s dynamic contractions preceded 6 min of intermittent arterial occlusions (5–10 s each, ∼250 mmHg) for k measurement. k was lower (P < 0.05) in COPD (1.43 ± 0.4 min−1; CV = 9.8 ± 5.9%, ICC = 0.88) than controls (1.74 ± 0.69 min−1; CV = 9.9 ± 8.4%; ICC = 0.93). Poor k reproducibility was more common when post-contraction mV̇O2 and deoxygenation were low, suggesting insufficient exercise intensity for mitochondrial activation and/or the NIRS signal contained little light reflected from active muscle. The NIRS assessment was well tolerated and reproducible for muscle dysfunction evaluation in COPD.