Due to the invasiveness of a muscle biopsy, there is fragmentary information on the existence and possible origin of a sexual dimorphism in the skeletal muscle concentrations of the energy delivery-related metabolites carnosine, creatine and carnitine. As these metabolites can be non-invasively monitored by proton magnetic resonance spectroscopy, this technique offers the possibility to investigate if sexual dimorphisms are present in an adult reference population and if these dimorphisms originated during puberty using a longitudinal design. Concentrations of carnosine, creatine and carnitine were examined using proton magnetic resonance spectroscopy in the soleus and gastrocnemius muscles of an adult reference population of female (n=50) and male adults (n=50). For the longitudinal follow-up over puberty, 29 boys and 28 girls were scanned pre-puberty. Six years later, 24 boys and 24 girls were rescanned post-puberty. A sexual dimorphism was present in carnosine and creatine, but not carnitine, in the adult reference population. Carnosine was 28.5% higher in the gastrocnemius (P<0.001) and carnosine and creatine were respectively 19.9% (P<0.001) and 18.2% (P<0.001) higher in the soleus of male, when compared to female adults. Through puberty, carnosine increased more in male subjects in comparison to female subjects, both in the gastrocnemius (+10.43 and -10.83%, respectively; interaction effect: P=0.002) and in the soleus (+24.30 and +5.49%, respectively; interaction effect: P=0.012). No significant effect of puberty was found in either creatine (interaction effect: P=0.307) or carnitine (interaction effect: P=0.066). A sexual dimorphism in the adult human muscle is present in carnosine and creatine, but not in carnitine.