This study examined the effects of prolonged exercise on human quadriceps muscle contractile function and homogenate sarcoplasmic reticulum Ca2+ uptake and Ca2+-adenosinetriphosphatase activity. Ten untrained men cycled at 75 +/- 2% (SE) peak oxygen consumption until exhaustion. Biopsies were taken from the right vastus lateralis muscle at rest, exhaustion, and 20 and 60 min postexercise. Peak tension and half relaxation time of the left quadriceps muscle were measured during electrically evoked twitch and tetanic contractions and a maximal voluntary isometric contraction at rest, exhaustion, and 10, 20, and 60 min postexercise. At exhaustion, homogenate Ca2+ uptake and Ca2+ adenosinetriphosphatase activity were reduced by 17 +/- 4 and 21 +/- 5%, respectively, and remained depressed after 60 min recovery (P </= 0.01). Muscle ATP, creatine phosphate, and glycogen were all depressed at exhaustion (P </= 0.01). Peak tension during a maximal voluntary contraction, a twitch, and a 10-Hz stimulation were reduced after exercise by 28 +/- 3, 45 +/- 6, 65 +/- 5%, respectively (P </= 0.01), but no slowing of half relaxation times were found. Thus fatigue induced by prolonged exercise reduced muscle Ca2+ uptake, but this did not cause a slower relaxation of evoked contractions.