We investigated the hypothesis that a program of prolonged endurance training, previously shown to decrease metabolic perturbations to acute exercise within 5 days of training, would result in greater metabolic adaptations after a longer training duration. Seven healthy male volunteers [O2 consumption = 3.52 +/- 0.20 (SE) l/min] engaged in a training program consisting of 2 h of cycle exercise at 59% of pretraining peak O2 consumption (VO2peak) 5-6 times/wk. Responses to a 90-min submaximal exercise challenge were assessed pretraining (PRE) and after 5 and 31 days of training. On the basis of biopsies obtained from the vastus lateralis muscle, it was found that, after 5 days of training, muscle lactate concentration, phosphocreatine (PCr) hydrolysis, and glycogen depletion were reduced vs. PRE (all P < 0.01). Further training (26 days) showed that, at 31 days, the reduction in PCr and the accumulation of muscle lactate was even less than at 5 days (P < 0.01). Muscle oxidative potential, estimated from the maximal activity of succinate dehydrogenase, was increased only after 31 days of training (+41%; P < 0.01). In addition, VO2peak was only increased (10%) by 31 days (P < 0.05). The results show that a period of short-term training results in many characteristic training adaptations but that these adaptations occurred before increases in mitochondrial potential. However, a further period of training resulted in further adaptations in muscle metabolism and muscle phosphorylation potential, which were linked to the increase in muscle mitochondrial capacity.