AbstractIn skeletal muscle, glycogen particles are distributed both within and between myofibrils, as well as just beneath the sarcolemma. Their precise localisation may influence their degradation rate. Here, we investigated how exercise at different intensities and durations (1‐ and 15‐min maximal exercise) with known variations in glycogenolytic rate and contribution from anaerobic metabolism affects utilisation of the distinct pools. Furthermore, we investigated how decreased glycogen availability achieved through lowering carbohydrate and energy intake after glycogen‐depleting exercise affect the storage of glycogen particles (size, numerical density, localisation). Twenty participants were divided into two groups performing either a 1‐min (n = 10) or a 15‐min (n = 10) maximal cycling exercise test. In a randomised, counterbalanced, cross‐over design, the exercise tests were performed following short‐term consumption of two distinct diets with either high or moderate carbohydrate content (10 vs. 4 g kg⁻¹ body mass (BM) day⁻¹) mediating a difference in total energy consumption (240 vs. 138 g kg⁻¹ BM day⁻¹). Muscle biopsies from m. vastus lateralis were obtained before and after the exercise tests. Intermyofibrillar glycogen was preferentially utilised during the 1‐min test, whereas intramyofibrillar glycogen was preferentially utilised during the 15‐min test. Lowering carbohydrate and energy intake after glycogen‐depleting exercise reduced glycogen availability by decreasing particle size across all pools and diminishing numerical density in the intramyofibrillar and subsarcolemmal pools. In conclusion, distinct subcellular glycogen pools were differentially utilised during 1‐min and 15‐min maximal cycling exercise. Additionally, lowered carbohydrate and energy consumption after glycogen‐depleting exercise altered glycogen storage by reducing particle size and numerical density, depending on subcellular localisation. imageKey points In human skeletal muscle, glycogen particles are localised in distinct subcellular compartments, referred to as intermyofibrillar, intramyofibrillar and subsarcolemmal pools. The intermyofibrillar and subsarcolemmal pools are close to mitochondria, while the intramyofibrillar pool is at a distance from mitochondria. We show that 1 min of maximal exercise is associated with a preferential utilisation of intermyofibrillar glycogen, and, on the other hand, that 15 min of maximal exercise is associated with a preferential utilisation of intramyofibrillar glycogen. Furthermore, we demonstrate that reduced glycogen availability achieved through lowering carbohydrate and energy intake after glycogen‐depleting exercise is characterised by a decreased glycogen particle size across all compartments, with the numerical density only diminished in the intramyofibrillar and subsarcolemmal compartments. These results suggest that exercise intensity influences the subcellular pools of glycogen differently and that the dietary content of carbohydrates and energy is linked to the size and subcellular distribution of glycogen particles.