The different bovine muscle fibre types I, IIA and IIX are characterised by their preferred metabolic pathway, either oxidative (I, IIA) or glycolytic (IIX), and their contraction speed, either slow-twitch (I) or fast-twitch (IIA, IIX). These physiological specificities are associated with variations in intracellular composition and their fluorescence spectra signatures. We hypothesised that these slight differences in autofluorescence responses could be used to discriminate the muscle fibre types by fluorescence imaging. Serial histological cross-sections of beef longissimus dorsi were performed: the start set was used to identify the metabolic and contractile type of muscle fibres by both immunohistoenzymology and immunohistofluorescence, and the following set was used to acquire synchrotron–deep ultraviolet (UV) autofluorescence images after excitation in the UV range (275 nm and 315 nm). This strategy made it possible to explore the label-free autofluorescence of muscle cells previously subtyped by histochemistry. Glycolytic cells (IIX) showed more intense fluorescence than oxidative cells (I and IIA) with near-90 % accuracy. This discrimination is more specifically assigned to the fluorescence of nicotinamide adenine dinucleotide. UV autofluorescence was unable to discriminate contractile type.