Contractile failure during various types of exercise has been attributed to intramuscular metabolic changes. We examined the temporal changes in force-generating capacity and metabolic state during intermittent isometric contractions in humans. One-legged quadriceps contractions at 30% maximum voluntary contraction (MVC) were executed for 6 s, with 4 s of rest between. The decrease in force-generating capacity was tested from brief MVC's and short bursts of 50-Hz stimulation applied at 5-min intervals. After 1 min of exercise, the MVC force declined linearly and in parallel to the 50-Hz stimulation force, indicating that the contractile failure was due to intramuscular processes. After 30 min of exercise the MVC force had declined by approximately 40% compared with the value obtained after 1 min. In separate experiments the same contraction protocol was followed, but two-legged contractions were used. Muscle biopsies taken after 5, 15, and 30 min of exercise showed only minor changes in the concentrations of glycogen, lactate, creatine phosphate (CrP), and ATP. However, at exhaustion, defined as loss of ability to sustain the target force, the concentrations of CrP and glycogen were reduced by 73 and 32%, and muscle lactate concentration had increased to 4.8 mmol/kg wet wt. Thus the gradual decline in force-generating capacity was not due to lactacidosis or lack of substrates for ATP resynthesis and must have resulted from excitation/contraction coupling failure, whereas exhaustion was closely related to phosphagen depletion, without significant lactacidosis.