We investigated the mechanisms of muscle fatigue in ALS. In the muscles of ALS patients and healthy control subjects, we examined (1) fatigue using measurements of muscle force, (2) energy metabolism using phosphorus-31 magnetic resonance spectroscopy, and (3) activation using neurophysiologic measures and MRI. During 25 minutes of intermittent isometric exercise of the tibialis anterior muscle, both maximum voluntary and tetanic force declined more in patients than in controls, indicating greater fatigability in ALS. There was a similar decline of voluntary and tetanic force, suggesting that much of the fatigue was not central. Evoked compound muscle action potential amplitudes were preserved during exercise in both groups, indicating no failure of neuromuscular transmission; this result suggests that the source of fatigue was not at the neuromuscular junction or within the muscle membrane. In spite of greater fatigability, changes during exercise in energy metabolites and proton signal intensity tended to be less in ALS patients compared with controls, suggesting impaired muscular activation. We conclude that the greater muscle fatigue in ALS patients results from activation impairment, due in part to alterations distal to the muscle membrane.