Purpose: Different strategies for near-infrared spectroscopy (NIRS)-derived muscle oxidative capacity assessment have been reported. This study compared and evaluated (I) approaches for averaging trials; (II) NIRS signals and blood volume correction equations; (III) the assessment of vastus lateralis (VL) and tibialis anterior (TA) muscles in two fitness levels groups. Methods: Thirty-six participants [18 chronically trained (CT: 14 males, 4 females) and 18 untrained (UT: 10 males, 8 females)] participated in this study. Two trials of twenty transient arterial occlusions were performed for NIRS-derived muscle oxidative capacity assessment. Muscle oxygen consumption ($$dotV$$VO2m) was estimated from deoxygenated hemoglobin (HHb), corrected for blood volume changes following Ryan (HHbR) and Beever (HHbB) equations, and from oxygen saturation (StO2) in VL and TA. Results: Superimposing or averaging $$dotV$$ VO2m or averaging the rate constants (k) from the two trials resulted in equivalent k values [two one-sided tests (TOST) procedure with 5% equivalence margin— P textless 0.001]. Whereas HHbR (2.35 ± 0.61 min−1) and HHbB (2.34 ± 0.58 min−1) derived k were equivalent (P textless 0.001), StO2 derived k (2.81 ± 0.92 min−1) was greater ( Ptextless 0.001) than both. k values were greater in CT vs UT in both muscles (VL: + 0.68 min−1, P = 0.002; TA: + 0.43 min−1, P = 0.01). Conclusion: Different approaches for averaging trials lead to similar . HHb and StO2 signals provided different k, although different blood volume corrections did not impact, k. Group differences in k were detected in both muscles.