The purpose of this study was to examine the effect of β-alanine (BA) ingestion on tissue carnosine levels and the impact such changes would have on combat specific activity. Eighteen soldiers (19.9 ± 0.8 year) from an elite combat unit were randomly assigned to either a BA or placebo (PL) group. Before and following a 30-day supplementation period carnosine content of the gastrocnemius muscle and brain was determined by proton magnetic resonance spectroscopy. During each testing session, participants performed military relevant tasks that included a 2.5 km run, a 1-min sprint, 50-m casualty carry, repeated 30-m sprints with target shooting, and a 2-min serial subtraction test (SST) to assess cognitive function under stressful conditions. A significant elevation (p = 0.048) in muscle carnosine content was noted in BA compared to PL. Changes in muscle carnosine content was correlated to changes in fatigue rate (r = 0.633, p = 0.06). No changes (p = 0.607) were observed in brain carnosine content. Following supplementation, no differences were noted in 2.5 km run, 1-min sprint, repeated sprint, or marksmanship performance, but participants in BA significantly (p = 0.044) improved their time for the 50-m casualty carry and increased their performance (p = 0.022) in the SST compared to PL. In summary, 30-days of BA ingestion can increase muscle carnosine content and improve aspects of military specific performance. Although cognitive performance was significantly greater in participants consuming BA compared to placebo, current study methods were unable to detect any change in brain carnosine levels, thus, the precise mechanism underlying these effects remains elusive.