["The Journal of Physiology, EarlyView. ", "\nAbstract figure legend The influence of haemoglobin–O2 affinity on aerobic capacity in hypoxia has been contentious. Many high‐altitude natives have greater haemoglobin–O2 affinity (lower P50, the O2 pressure at 50% haemoglobin saturation) than their low‐altitude counterparts, but the advantages of this change for aerobic metabolism have often remained unresolved. We examined this issue in deer mice (Peromyscus maniculatus) native to high altitude. Our findings suggest that the optimal haemoglobin–O2 affinity is greater in high‐altitude mice than in low‐altitude mice, potentially resulting from a greater capacity to extract and consume O2 in active tissues.\n\n\n\n\n\n\n\n\n\nAbstract\nHigh‐altitude hypoxia constrains tissue O2 supply, but several high‐altitude populations have evolved adaptations to overcome this challenge. Evolved increases in haemoglobin–O2 (Hb‐O2) affinity are pervasive across high‐altitude taxa, but the influence of such increases on aerobic capacity in hypoxia remains contentious. The influence of Hb‐O2 affinity could depend on the capacity to extract O2 from the blood, but this possibility is poorly understood. We examined this issue in deer mice (Peromyscus maniculatus), which are found from sea level to >4300 m elevation in the Rocky Mountains. Mice from populations native to high and low altitudes were born and raised in captivity. Low‐altitude mice were acclimated to warm (25°C) normoxia and high‐altitude mice were acclimated to cold (5°C) hypoxia (∼12 kPa O2), creating two groups with distinct capacities for O2 transport. Aerobic capacity for thermogenesis was measured in hypoxia after each of three pharmacological treatments: saline (control), efaproxiral (decreases Hb‐O2 affinity) and cyanate (increases Hb‐O2 affinity). High‐altitude mice had greater aerobic capacity in hypoxia, in association with higher arterial O2 saturation (SaO2${{S}_{{\\mathrm{a}}{{{\\mathrm{O}}}_2}}}$) and lower P50 (O2 pressure at 50% Hb saturation) in most conditions. The P50 at which aerobic capacity was greatest was lower in high‐altitude mice than in low‐altitude mice. High‐altitude mice also had greater uncoupling protein 1 (UCP‐1) content in brown adipose tissue and greater cytochrome oxidase activity in gastrocnemius muscle. These results suggest that optimal Hb‐O2 affinity and SaO2${{S}_{{\\mathrm{a}}{{{\\mathrm{O}}}_2}}}$ are greater in high‐altitude mice, in association with a greater capacity to extract and consume O2 in thermogenic tissues.\n\n\n\n\n\n\n\n\n\nKey points\n\nEvolved increases in haemoglobin–O2 affinity are pervasive across high‐altitude taxa, but the influence of such increases on aerobic capacity in hypoxia remains contentious.\nWe examined whether the influence of haemoglobin–O2 affinity on aerobic capacity for thermogenesis is altered in high‐altitude deer mice.\nUsing pharmacological treatments to manipulate haemoglobin–O2 affinity, we found that aerobic capacity in hypoxia was greatest at higher affinities in high‐altitude mice than in low‐altitude mice.\nSkeletal muscle and brown adipose tissue had more oxidative and thermogenic phenotypes in high‐altitude mice.\nThese results suggest that the optimal haemoglobin–O2 affinity in hypoxia is greater in high‐altitude deer mice, potentially resulting from a greater capacity to extract and consume O2 in active tissues.\n\n\n"]