This review describes in vivo NMR measurements of metabolite diffusion. NMR spectroscopy can employ a variety of endogenous marker molecules to obtain information on various aspects of in vivo biochemistry and biophysics from the diffusion characteristics of these markers. NMR-based metabolite diffusion data give an insight into the physico-chemical properties of the intracellular compartment and of changes therein in relation to development and pathology. Recent applications have proven that in vivo diffusion spectroscopy provides exciting opportunities to both fundamental and applied research.