Mitochondrial diseases are a clinically heterogeneous group of disorders related to dysfunction of various components of oxidative metabolism. Common manifestations of these diseases include encephalopathy, skeletal myopathy, and cardiomyopathy, but essentially any tissue can be affected. To understand better the pathogenesis of mitochondrial disease and to potentially evaluate novel therapies, several mouse models have been reported in the literature over the past few years. In assessing genetically altered mice as potential models of human mitochondrial disease, careful behavioral and physiologic analyses are essential components of the overall phenotypic characterization. Noninvasive, in vivo approaches are useful because they assess end-organ and multiorgan function in a whole-organism context, as well as permit serial measurements of individual animals over time. This review presents various behavioral and exercise physiology protocols that can be used for the evaluation of potential mouse models of human mitochondrial disorders.