The oxygen-dependent processes of ATP synthesis in mitochondria is same in all mammals and birds (endothermic animals). However, some endothermic species have higher maximum O₂ consumption rate per unit mass (VO₂ max/Mb) than others during aerobic exercise. It is unclear whether the mitochondria of these endothermic species achieving higher VO₂ max/Mb have inherently higher biochemical capacities for respiration than mitochondria from other species. To understand the relation between functional capacities (biochemical capacity) and maximum physiological requirements, the respiration rates of flight muscle mitochondria of hovering C. anna (in vivo mVO₂) and the maximum oxygen consumption rate of isolated mitochondria (in vitro mVO₂) were compared. To measure whole body VO₂max of C. anna, C. anna hovered in heliox (21% oxygen and 79% helium gas mixture). In vivo mVO₂ of C. anna flight muscles calculated using whole body VO₂max was 10.5 ml O₂/min x ml on average. Since in vitro mVO₂ values are substrate-dependent, in vitro mVO₂ was measured using different combinations of substrates: ones that donate electrons to the electron transport chain via NADH and those that donate electrons via both NADH and FADH2 (Gnaiger, 2009). The NADH and FADH2 -linked substrate combination elicited the highest in vitro mVO₂ which was 6.8 ml O₂/min × ml on average. The in vitro mVO₂ measurement methods used in this study brought a gap between in vivo and in vitro rates closer than the previous study (Suarez et al., 1990); however, biochemical capacity was still 17.3 % lower than the maximum physiological rate. C. anna flight muscle cytochrome aa3 content measured using spectrophotometric method was 83.88 n mol/g muscle. The electron flux through cytochrome aa3 calculated using cytochrome aa3 content and VO₂ of C. anna hovering in heliox at 86.7 electrons/second. This electron flux rate is only slightly higher than the rate of rat heart, but cytochrome aa3 content per gram muscle is about 6 times higher in hummingbird flight muscle than the rat heart muscle. This indicates the level of cytochrome aa3 expression and the physiological requirements are matched to maximum requirements in both hummingbird pectoral and rat cardiac muscles.