["Acta Physiologica, Volume 231, Issue 4, April 2021. ", "\nAbstract\n\nAim\nThis study sought to provide a statistically robust reference for measures of mitochondrial function from standardized high‐resolution respirometry with permeabilized human skeletal muscle (ex vivo), compare analogous values obtained via indirect calorimetry, arterial‐venous O2 differences and 31P magnetic resonance spectroscopy (in vivo) and attempt to resolve differences across complementary methodologies as necessary.\n\n\nMethods\nData derived from 831 study participants across research published throughout March 2009 to November 2019 were amassed to examine the biological relevance of ex vivo assessments under standard conditions, ie physiological temperatures of 37°C and respiratory chamber oxygen concentrations of ~250 to 500 μmol/L.\n\n\nResults\nStandard ex vivo‐derived measures are lower (Z ≥ 3.01, P ≤ .0258) en masse than corresponding in vivo‐derived values. Correcting respiratory values to account for mitochondrial temperatures 10°C higher than skeletal muscle temperatures at maximal exercise (~50°C): (i) transforms data to resemble (Z ≤ 0.8, P > .9999) analogous yet context‐specific in vivo measures, eg data collected during maximal 1‐leg knee extension exercise; and (ii) supports the position that maximal skeletal muscle respiratory rates exceed (Z ≥ 13.2, P < .0001) those achieved during maximal whole‐body exercise, e.g. maximal cycling efforts.\n\n\nConclusion\nThis study outlines and demonstrates necessary considerations when actualizing the biological relevance of human skeletal muscle respiratory control, metabolic flexibility and bioenergetics from standard ex vivo‐derived assessments using permeabilized human muscle. These findings detail how cross‐procedural comparisons of human skeletal muscle mitochondrial function may be collectively scrutinized in their relationship to human health and lifespan.\n\n"]