Until now, the studies focused on mitochondrial ROS production have essentially reported on basal non-phosphorylating and maximal phosphorylating states. Such double assessment is useful to compare species, populations or individuals facing environment variations but does not fully reflect the interplay between oxygen consumption and ROS formation, particularly at intermediate activity levels. ROS generation remains low until a threshold value of mitochondrial activity is reached, at which point this production abruptly increases. On the whole, the variation of mitochondrial thresholds for H2O2 generation is globally unknown in animals. The aim of the present study was to i) evaluate the interplay between mitochondrial respiration and H2O2 release during the transition from basal to maximal phosphorylating states in different mammals’ skeletal muscle mitochondria in presence of a mixture of substrates (pyruvate, malate and succinate), ii) estimate the range of variation of the resulting threshold values and iii) assess the allometry of these parameters in mammal species ranging from 5 g to 500 kg. Our results showed a substrate-dependent allometric relationship, with small mammals having higher mitochondrial radical production than larger ones (except for the smallest species, Mus mattheyi). Despite these interspecific differences, all species exhibited identical burst of H2O2 release at a low rate of oxidative phosphorylation, essentially once the activity of mitochondrial oxidative phosphorylation reached 26% of the maximal respiration. This identical value in every species studied suggests that the threshold for H2O2 generation is a general characteristic of skeletal muscle mitochondria in mammalian species.