AbstractGlycogen particles are situated in key areas of the muscle cell in the vicinity of the main energy‐consumption sites and may be utilised heterogeneously dependent on the nature of the metabolic demands. The present study aimed to investigate the time course of fibre type‐specific utilisation of muscle glycogen in three distinct subcellular fractions (intermyofibrillar, IMF; intramyofibrillar, Intra; and subsarcolemmal, SS) during repeated high‐intensity intermittent exercise. Eighteen moderately to well‐trained male participants performed three periods of 10 × 45 s cycling at ∼105% watt max (EX1–EX3) coupled with 5 × 6 s maximal sprints at baseline and after each period. Muscle biopsies were sampled at baseline and after EX1 and EX3. A higher glycogen breakdown rate in type 2 compared to type 1 fibres was found during EX1 for the Intra (−72 vs. −45%) and IMF (−59 vs. −35%) glycogen fractions (P < 0.001) but with no differences for SS glycogen (−52 vs. −40%). In contrast, no fibre type differences were observed during EX2–EX3, where the utilisation of Intra and IMF glycogen in type 2 fibres was reduced, resulting in depletion of all three subcellular fractions to very low levels post‐exercise within both fibre types. Importantly, large heterogeneity in single‐fibre glycogen utilisation was present with an early depletion of especially Intra glycogen in individual type 2 fibres. In conclusion, there is a clear fibre type‐ and localisation‐specific glycogen utilisation during high‐intensity intermittent exercise, which varies with time course of exercise and is characterised by exacerbated pool‐specific glycogen depletion at the single‐fibre level. imageKey points Muscle glycogen is the major fuel during high‐intensity exercise and is stored in distinct subcellular areas of the muscle cell in close vicinity to the main energy consumption sites. In the present study quantitative electron microscopy imaging was used to investigate the utilisation pattern of three distinct subcellular muscle glycogen fractions during repeated high‐intensity intermittent exercise. It is shown that the utilisation differs dependent on fibre type, subcellular localisation and time course of exercise and with large single‐fibre heterogeneity. These findings expand on our understanding of subcellular muscle glycogen metabolism during exercise and may help us explain how reductions in muscle glycogen can attenuate muscle function even at only moderately lowered whole‐muscle glycogen concentrations.