Key points: Muscle glycogen content is associated with muscle function, but the physiological link between the two is poorly understood. This study investigated the effects of inhibiting glycogenolysis, while maintaining high overall energy status, on different aspects of muscle function. We demonstrate here that Na +,K +-ATPase activity depends on glycogenolytically derived ATP regardless of high global ATP, with a decrease in activity leading to reduced force production and accelerated fatigue development. The results support the concept of compartmentalized energy transfer with glycogen metabolism playing a crucial role in intramuscular ATP resynthesis and ion regulation. This study gives specific insights into muscular function and may help towards a better understanding of glycogen storage diseases and muscle fatigue. Abstract: Skeletal muscle glycogen content is associated with muscle function and fatigability. However, little is known about the physiological link between glycogen content and muscle function. Here we aimed to investigate the importance of glycogenolytically derived ATP per se on muscle force and action potential (AP) repriming period, i.e. the time before a second AP can be produced (indicative of Na +,K +-ATPase activity). Single fibres from rat extensor digitorum longus muscles were isolated and mechanically skinned in order to investigate force production and the AP repriming period while global ATP and PCr concentrations were kept high. The importance of glycogenolytically derived ATP was studied by inhibition of glycogen phosphorylase (1,4-dideoxy-1,4-imino-d-arabinitol (DAB; 2 mm) or CP-316,819 (CP; 10 µm)) or glycogen removal (amyloglucosidase, 20 U ml −1). Tetanic force decreased by (mean (SD)) 21 (15)% (P < 0.001) and 76 (28)% (DAB) or 94 (6)% (CP, P < 0.001) in well-polarized and partially depolarized fibres, respectively. In depolarized fibres, twitch force decreased by 16 (10)% and 55 (26)% with DAB and CP, respectively, with no effect in well-polarized fibres (84 (10)%, P = 0.14). There was no effect of glycogen phosphorylase inhibition on repriming period in well-polarized fibres (median (25th, 75th percentile): 5 (4, 5) vs. 4 (4, 5) ms, P = 0.26), while the repriming period was prolonged from 6 (5, 7) to 8 (7, 10) ms (P = 0.01) in partially depolarized fibres. In line with this, glycogen removal increased repriming period from 5 (5, 6) to 6 (5, 7) ms (P = 0.003) in depolarized fibres. Together, these data strongly indicate that blocking glycogenolysis attenuates Na +,K +-ATPase activity, which in turn increases the repriming period and reduces force, demonstrating a functional link between glycogenolytically derived ATP and force production.