Abstract Aims: Mitochondria are responsible for meeting the majority of the energetic demand of most tissues. They also play a major role in regulating cell survival. These dual roles of mitochondria place them at the centre of many pathologies leading to tissue degeneration and disruption of energy balance. The prominent role of mitochondria in ageing and disease has led to a tremendous growth in mitochondrial research at the cellular and molecular level. We describe below a new non-invasive approach to measure mitochondrial function that will bridge the gap between our understanding of mitochondrial function in vitro and that in the intact organism. Methods and Results: This approach uses optical and magnetic resonance spectroscopy to measure in vivo O2 consumption and ATP synthesis rates, respectively, from skeletal muscle. These values lead to a quantitative assessment of the mitochondrial ATP/O2 or P/O. The P/O represents the efficiency of coupling between phosphorylation and oxygen consumption in the mitochondria, which is a measure of mitochondrial dysfunction. Conclusions: This work represents a significant advance in research on the role of mitochondria in degenerative disease and ageing because it allows a quantitative measure of mitochondrial pathology in vivo. The non-invasive nature of this approach also enables repeated measures of mitochondrial function on the same individual, thereby making this a potentially useful diagnostic technique. The results from this work have led to insights into the coupling of ATP synthesis to oxidation and the regulation of oxidative phosphorylation by intracellular PO2.