Altered metabolic activity contributes to the pathogenesis of a number of diseases, including diabetes, heart failure, cancer, fibrosis and neurodegeneration. These diseases, and organismal metabolism more generally, are only partially recapitulated by cell culture models. Accordingly, it is important to measure metabolism in vivo. Over the past century, researchers studying glucose homeostasis have developed strategies for the measurement of tissue-specific and whole-body metabolic activity (pathway fluxes). The power of these strategies has been augmented by recent advances in metabolomics technologies. Here, we review techniques for measuring metabolic fluxes in intact mammals and discuss how to analyse and interpret the results. In tandem, we describe important findings from these techniques, and suggest promising avenues for their future application. Given the broad importance of metabolism to health and disease, more widespread application of these methods holds the potential to accelerate biomedical progress.