["The Journal of Physiology, EarlyView. ", "\nAbstract figure legend This graphical abstract illustrates a novel, non‐invasive approach to quantify mitochondrial oxygen (O2) affinity in vivo in human skeletal muscle using 1H magnetic resonance spectroscopy (1H‐MRS) of deoxymyoglobin (dMb). During brief arterial occlusion, dMb kinetics were used to simultaneously estimate intracellular oxygen tension (i PO2${{P}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$) and mitochondrial oxygen consumption (V̇O2${{\\dot{V}}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$) in the plantar flexor muscles. The relationship between V̇O2${{\\dot{V}}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$ and i PO2${{P}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$ enabled the determination of the apparent mitochondrial P50 for O2, revealing a very high mitochondrial O2 affinity (P50 = 0.5 mmHg). These findings indicate that resting human skeletal muscle operates with a large O2 reserve, well above the range of mitochondrial O2 limitation.\n\n\n\n\n\n\n\n\n\nAbstract\nMitochondrial oxygen (O2) affinity is a fundamental determinant of oxidative phosphorylation capacity, which has yet to be directly measured in human skeletal muscle in vivo. To determine the apparent mitochondrial O2 affinity (i.e. P50) of the skeletal muscle, we used proton‐magnetic resonance spectroscopy (1H‐MRS) of deoxymyoglobin (dMb) during a circulatory occlusion of the lower limb to simultaneously quantify intracellular partial pressure of O2 (i PO2${{P}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$) and resting tissue‐specific O2 consumption (V̇O2${{\\dot{V}}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$) in the gastrocnemius muscle of sedentary young adults. Under these resting conditions, the V̇O2${{\\dot{V}}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$–i PO2${{P}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$ relationship conformed to a Michaelis–Menten kinetics model (goodness of fit: r2 = 0.84 ± 0.13). The estimated Vss, reflecting basal metabolic rate, reached 0.20 ± 0.06 mM min−1, and the apparent mitochondrial P50 was 0.50 ± 0.38 mmHg. Vss and P50 were positively correlated (r = 0.85, P = 0.0009). This strong correlation remained after log transformation (r = 0.82, P = 0.0020). These results, obtained in human muscles in vivo, demonstrate that mitochondrial respiration exhibits a very high O2 affinity (P50 ≈ 0.5 mmHg). In addition, it quantitatively identifies the range across which oxidative phosphorylation in the skeletal muscle is O2‐independent (resting i PO2${{P}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$ under physiological conditions) or becomes O2‐sensitive (severe exercise and hypoxia). Moreover, the finding that muscle respiratory flux was associated with the apparent mitochondrial O2 affinity suggests additional regulatory mechanisms within the respiratory chain to fine‐tune oxidative phosphorylation to muscle ATP demand even at rest. This study provides a robust quantitative framework for interpreting in vivo respiratory control in both health and disease.\n\n\n\n\n\n\n\n\n\nKey points\n\nMitochondrial oxygen affinity (P50) is central to oxidative phosphorylation, yet has not been directly quantified in human skeletal muscle in vivo.\nUsing deoxymyoglobin 1H‐MRS during 8 min of lower‐limb ischaemia, we simultaneously measure intracellular partial pressure of O2 (i PO2${{P}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$) and resting tissue‐specific O2 consumption (V̇O2${{\\dot{V}}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$) in the resting gastrocnemius of healthy young adults.\nThe V̇O2${{\\dot{V}}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$–PO2${{P}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$ relationship followed Michaelis–Menten kinetics, yielding a Vss of 0.20 mM min−1 and an apparent mitochondrial P50 of 0.50 mmHg, indicating very high O2 affinity.\nResting apparent mitochondrial P50 was far below previously reported i PO2${{P}_{{{{\\mathrm{O}}}_{\\mathrm{2}}}}}$ (∼34 mmHg), indicating a wide O2‐independent range and identifying conditions (severe hypoxia, high‐intensity exercise) in which oxidative phosphorylation becomes O2‐sensitive.\nVss and P50 were positively correlated (r = 0.85), suggesting in vivo coupling between respiratory flux and oxygen affinity and providing a potential quantitative framework for interpreting mitochondrial respiratory control in health and disease.\n\n\n"]