Most research on creatine has focused on short-term creatine loading and its effect on high-intensity performance capacity. Some studies have investigated the effect of prolonged creatine use during strength training. However, studies on the effects of prolonged creatine supplementation are lacking. In the present study, we have assessed the effects of both creatine loading and prolonged supplementation on muscle creatine content, body composition, muscle and whole-body oxidative capacity, substrate utilization during submaximal exercise, and on repeated supramaximal sprint, as well as endurance-type time-trial performance on a cycle ergometer. Twenty subjects ingested creatine or a placebo during a 5-day loading period (20 g.day(-1)) after which supplementation was continued for up to 6 weeks (2 g.day(-1)). Creatine loading increased muscle free creatine, creatine phosphate (CrP) and total creatine content ( P <0.05). The subsequent use of a 2 g.day(-1) maintenance dose, as suggested by an American College of Sports Medicine Roundtable, resulted in a decline in both the elevated CrP and total creatine content and maintenance of the free creatine concentration. Both short- and long-term creatine supplementation improved performance during repeated supramaximal sprints on a cycle ergometer. However, whole-body and muscle oxidative capacity, substrate utilization and time-trial performance were not affected. The increase in body mass following creatine loading was maintained after 6 weeks of continued supplementation and accounted for by a corresponding increase in fat-free mass. This study provides definite evidence that prolonged creatine supplementation in humans does not increase muscle or whole-body oxidative capacity and, as such, does not influence substrate utilization or performance during endurance cycling exercise. In addition, our findings suggest that prolonged creatine ingestion induces an increase in fat-free mass.