We investigated the role of limitations in aerobic metabolism, glycolysis and membrane excitability for development of high-frequency fatigue in isolated rat soleus. Muscles mounted on force transducers were incubated in buffer bubbled with 5% CO₂ and either 95% O₂ (oxygenated) or 95% N₂ (anoxic) and stimulated at 60 Hz continuously for 30-120 s or intermittently for 120 s. Cyanide (2 mM) and 2-deoxyglucose (10 mM) were used to inhibit aerobic metabolism and both glycolysis and aerobic metabolism, respectively. Excitability was reduced by carbacholine (10 µM), an nAChR agonist, or ouabain (10 µM), a Na⁺,K⁺-pump inhibitor. Membrane excitability was measured by recording M waves. Intracellular Na⁺,K⁺ contents and membrane potentials were measured using flame photometry and microelectrodes, respectively. During 120 s of continuous stimulation, oxygenated and anoxic muscles showed the same force loss. In oxygenated muscles, cyanide did not alter force loss for up to 90 s, whereas 2-deoxyglucose increased force loss (by 19-69%, P<0.01) from 14 s of stimulation. In oxygenated muscles, 60 s of stimulation reduced force, M wave area and amplitude by 70-90% (P<0.001). Carbacholine or ouabain increased intracellular Na⁺ content (P<0.001), induced a 7-8 mV membrane depolarisation (P<0.001), and accelerated the rate of force loss (by 250-414%) during 30 s of stimulation (P<0.001). Similar effects were seen with intermittent stimulation. In conclusion, limitations in glycolysis and subsequently also aerobic metabolism, as well as membrane excitability, but not aerobic metabolism alone, appear to play an important role in the development of high-frequency fatigue in isolated rat soleus muscle.