Substantial ATP supply by glycolysis is thought to reflect cellular anoxia in vertebrate muscle. An alternative hypothesis is that the lactate generated during contraction reflects sustained glycolytic ATP supply under well-oxygenated conditions. We distinguished these hypotheses by comparing intracellular glycolysis during anoxia to lactate efflux from muscle during sustained, aerobic contractions. We examined the tailshaker muscle of the rattlesnake because of its uniform cell properties, exclusive blood circulation, and ability to sustain rattling for prolonged periods. Here we show that glycolysis is independent of the O2 level and supplies one-third of the high ATP demands of sustained tailshaking. Fatigue is avoided by rapid H+ and lactate efflux resulting from blood flow rates that are among the highest reported for vertebrate muscle. These results reject the hypothesis that glycolysis necessarily reflects cellular anoxia. Instead, they demonstrate that glycolysis can provide a high and sustainable supply of ATP along with oxidative phosphorylation without muscle fatigue.