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The final steps of the O2 cascade during exercise depend on the product of the microvascular‐to‐intramyocyte PO2${P}_{{{\rm{O}}}_{\rm{2}}}$ difference and muscle O2 diffusing capacity (DmO2$D{{\rm{m}}}_{{{\rm{O}}}_2}$). Non‐invasive methods to determine DmO2$D{{\rm{m}}}_{{{\rm{O}}}_2}$ in humans are currently unavailable. Muscle oxygen uptake (mV̇O2${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$) recovery rate constant (k), measured by near‐infrared spectroscopy (NIRS) using intermittent arterial occlusions, is associated with muscle oxidative capacity in vivo. We reasoned that k would be limited by DmO2$D{{\rm{m}}}_{{{\rm{O}}}_2}$ when muscle oxygenation is low (kLOW), and hypothesized that: (i) k in well oxygenated muscle (kHIGH) is associated with maximal O2 flux in fibre bundles; and (ii) ∆k (kHIGH – kLOW) is associated with capillary density (CD). Vastus lateralis k was measured in 12 participants using NIRS after moderate exercise. The timing and duration of arterial occlusions were manipulated to maintain tissue saturation index within a 10% range either below (LOW) or above (HIGH) half‐maximal desaturation, assessed during sustained arterial occlusion. Maximal O2 flux in phosphorylating state was 37.7 ± 10.6 pmol s−1 mg−1 (∼5.8 ml min−1 100 g−1). CD ranged 348 to 586 mm–2. kHIGH was greater than kLOW (3.15 ± 0.45 vs. 1.56 ± 0.79 min–1, P < 0.001). Maximal O2 flux was correlated with kHIGH (r = 0.80, P = 0.002) but not kLOW (r = –0.10, P = 0.755). Δk ranged –0.26 to –2.55 min–1, and correlated with CD (r = –0.68, P = 0.015). mV̇O2${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$k reflects muscle oxidative capacity only in well oxygenated muscle. ∆k, the difference in k between well and poorly oxygenated muscle, was associated with CD, a mediator of DmO2$D{{\rm{m}}}_{{{\rm{O}}}_2}$. Assessment of muscle k and ∆k using NIRS provides a non‐invasive window on muscle oxidative and O2 diffusing capacity. Key points: We determined post‐exercise recovery kinetics of quadriceps muscle oxygen uptake (mV̇O2${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$) measured by near‐infrared spectroscopy (NIRS) in humans under conditions of both non‐limiting (HIGH) and limiting (LOW) O2 availability, for comparison with biopsy variables.The mV̇O2${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ recovery rate constant in HIGH O2 availability was hypothesized to reflect muscle oxidative capacity (kHIGH) and the difference in k between HIGH and LOW O2 availability (∆k) was hypothesized to reflect muscle O2 diffusing capacity.kHIGH was correlated with phosphorylating oxidative capacity of permeabilized muscle fibre bundles (r = 0.80).∆k was negatively correlated with capillary density (r = −0.68) of biopsy samples.NIRS provides non‐invasive means of assessing both muscle oxidative and oxygen diffusing capacity in vivo. [ABSTRACT FROM AUTHOR]

Keywords: NIRS; muscle oxidative capacity; reliability; O2 diffusing capacity EN NIRS muscle oxidative capacity reliability O2 diffusing capacity 5165 5166 2 12/02/22 20221201 NES 221201 We thank Dr Manferdelli and colleagues for their Letter to the Editor (Manferdelli et al., [2]) regarding our recent work (Pilotto et al., [4]). We welcome the discussion around the novel approach we proposed for measurement of muscle oxidative capacity and muscle O SB 2 sb diffusion. Therefore, we concluded that the NIRS-based protocol represents a cost-effective and non-invasive means of assessing I in vivo i both muscle oxidative capacity and muscle O SB 2 sb diffusive capacity. [Extracted from the article]