Mitochondrial changes are at the centre of a wide range of maladies, including diabetes, neurodegeneration and ageing-related dysfunctions. Here we describe innovative optical and magnetic resonance spectroscopic methods that non-invasively measure key mitochondrial fluxes, ATP synthesis and O(2) uptake, to permit the determination of mitochondrial coupling efficiency in vivo (P/O: half the ratio of ATP flux to O(2) uptake). Three new insights result. First, mitochondrial coupling can be measured in vivo with the rigor of a biochemical determination and provides a gold standard to define well-coupled mitochondria (P/O approximately 2.5). Second, mitochondrial coupling differs substantially among muscles in healthy adults, from values reflective of well-coupled oxidative phosphorylation in a hand muscle (P/O = 2.7) to mild uncoupling in a leg muscle (P/O = 2.0). Third, these coupling differences have an important impact on cell ageing. We found substantial uncoupling and loss of cellular [ATP] in a hand muscle indicative of mitochondrial dysfunction with age. In contrast, stable mitochondrial function was found in a leg muscle, which supports the notion that mild uncoupling is protective against mitochondrial damage with age. Thus, greater mitochondrial dysfunction is evident in muscles with higher type II muscle fibre content, which may be at the root of the preferential loss of type II fibres found in the elderly. Our results demonstrate that mitochondrial function and the tempo of ageing varies among human muscles in the same individual. These technical advances, in combination with the range of mitochondrial properties available in human muscles, provide an ideal system for studying mitochondrial function in normal tissue and the link between mitochondrial defects and cell pathology in disease.