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Key points Muscle glycogen (the storage form of glucose) is consumed during muscle work and the depletion of glycogen is thought to be a main contributor to muscle fatigue. In this study, we used a novel approach to first measure fatigue-induced reductions in force and tetanic Ca2+ in isolated single mouse muscle fibres following repeated contractions and subsequently quantify the subcellular distribution of glycogen in the same fibre. Using this approach, we investigated whether the decreased tetanic Ca2+ induced by repeated contractions was associated with glycogen depletion in certain subcellular regions. The results show a positive correlation between depletion of glycogen located within the myofibrils and low tetanic Ca2+ after repetitive stimulation. We conclude that subcellular glycogen depletion has a central role in the decrease in tetanic Ca2+ that occurs during repetitive contractions. In skeletal muscle fibres, glycogen has been shown to be stored at different subcellular locations: (i) between the myofibrils (intermyofibrillar); (ii) within the myofibrils (intramyofibrillar); and (iii) subsarcolemmal. Of these, intramyofibrillar glycogen has been implied as a critical regulator of sarcoplasmic reticulum Ca2+ release. The aim of the present study was to test directly how the decrease in cytoplasmic free Ca2+ ([Ca2+](i)) during repeated tetanic contractions relates to the subcellular glycogen distribution. Single fibres of mouse flexor digitorum brevis muscles were fatigued with 70Hz, 350ms tetani given at 2s (high-intensity fatigue, HIF) or 10s (low-intensity fatigue, LIF) intervals, while force and [Ca2+](i) were measured. Stimulation continued until force decreased to 30% of its initial value. Fibres were then prepared for analyses of subcellular glycogen distribution by transmission electron microscopy. At fatigue, tetanic [Ca2+](i) was reduced to 70 +/- 4% and 54 +/- 4% of the initial in HIF (P<0.01, n=9) and LIF (P<0.01, n=5) fibres, respectively. At fatigue, the mean inter- and intramyofibrillar glycogen content was 60-75% lower than in rested control fibres (P<0.05), whereas subsarcolemmal glycogen was similar to control. Individual fibres showed a good correlation between the fatigue-induced decrease in tetanic [Ca2+](i) and the reduction in intermyofibrillar (P=0.051) and intramyofibrillar (P=0.0008) glycogen. In conclusion, the fatigue-induced decrease in tetanic [Ca2+](i), and hence force, is accompanied by major reductions in inter- and intramyofibrillar glycogen. The stronger correlation between decreased tetanic [Ca2+](i) and reduced intramyofibrillar glycogen implies that sarcoplasmic reticulum Ca2+ release critically depends on energy supply from the intramyofibrillar glycogen pool.

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