Carnosine (-alanyl-L-histidine) is present in high concentrations in human skeletal muscle. The ingestion of -alanine, the rate-limiting precursor of carnosine, has been shown to elevate the muscle carnosine content. We aimed to investigate, using proton magnetic resonance spectroscopy (proton MRS), whether oral supplementation with -alanine during 4 weeks would elevate the calf muscle carnosine content and affect exercise performance in 400m sprint-trained competitive athletes. Fifteen male athletes participated in a placebo-controlled, double-blind study and were supplemented orally for 4 weeks with either 4.8g/day -alanine or placebo. Muscle carnosine concentration was quantified in soleus and gastrocnemius by proton MRS. Performance was evaluated by isokinetic testing during 5 bouts of 30 maximal voluntary knee extensions, by endurance during isometric contraction at 45% MVC and by the indoor 400m running time. -Alanine supplementation significantly increased the carnosine content in both the soleus (+47%) and gastrocnemius (+37%). In placebo, carnosine remained stable in soleus while a small and significant increase of +16% occurred in gastrocnemius. Dynamic knee extension torque during the fourth and fifth bout was significantly improved with -alanine but not with placebo. Isometric endurance and 400m race time were not affected by treatment. In conclusion, 1) proton MRS can be used to non-invasively quantify human muscle carnosine content; 2) muscle carnosine is increased by oral β-alanine supplementation in sprint-trained athletes; 3) carnosine loading slightly but significantly attenuated fatigue in repeated bouts of exhaustive dynamic contractions; 4) the increase in muscle carnosine did not improve isometric endurance or 400m race time.