Skeletal muscle carnosine content can be increased via chronic beta-alanine ingestion, but the maximum increase achievable with supplementation is unknown. Additionally, the effects of prolonged supplementation on carnosine-related gene expression in humans are not known. Since athletes are likely to supplement for extended periods of time, determination of the long-term effects of beta-alanine supplementation is warranted. PURPOSE: To investigate the effects of 24-weeks of beta-alanine supplementation on muscle carnosine content and expression of genes related to carnosine metabolism. METHODS: Twenty-four males were supplemented with 6.4 g·day-1 of sustained release beta-alanine (BA; N=15) or placebo (PL; N=9) for 24 weeks. Every 4 weeks participants provided a muscle biopsy from the m. vastus lateralis, which was subsequently analysed for muscle carnosine content and gene expression (CARNS, TauT, ABAT, CNDP2, PHT1, PEPT2, PAT1). RESULTS: Carnosine content was increased from baseline at every time point in BA (all P<0.0001; Week 4: +11.4±7.0 mmol·kg-1dm, Week 8: +13.9±7.8 mmol·kg-1dm, Week 12: +17.0±8.6 mmol·kg-1dm, Week 16: +17.6±8.4 mmol·kg-1dm, Week 20: +21.2±7.9 mmol·kg-1dm, Week 24: +20.2±7.6 mmol·kg-1dm), but not PL (all P>0.05). Maximal changes ranged from +17.1 to +41.3 mmol·kg-1dm, and absolute maximal content ranged from 31.8 to 63.9 mmol·kg-1dm. There was an effect of supplement (P=0.002) on TauT with lower expression in BA (-36%, -39%, -27%, -57%, -46% and -35% at Weeks 4, 8, 12, 16, 20 and 24); no further differences in gene expression were shown. CONCLUSION: Twenty-four weeks of beta-alanine supplementation increased muscle carnosine content in all individuals at all time points, although absolute maximal changes were variable. Downregulation of the beta-alanine transporter TauT suggests it plays an important role in muscle carnosine accumulation with beta-alanine supplementation. These data demonstrate that individuals who supplement with beta-alanine for prolonged periods can maintain elevated muscle content throughout supplementation, despite downregulation of beta-alanine transporter expression.