This study analyzed the acute metabolic and mechanical responses to a specific repeated sprint ability (RSA) test. Eighteen male professional soccer players from a team of the First Division of Spanish National League participated. A 12 × 30-m RSA test with 30-second recovery together with countermovement jump test (CMJ) pre a post RSA test was performed. Mechanical responses (i.e., height performance in CMJ and speed loss) and metabolic responses (i.e., blood lactate and ammonia concentrations) were measured before and after exercise. A related sample t-test was used to analyze CMJ height pre-post changes as well as to compare pre- and post-exercise lactate and ammonia levels. Countermovement jump height loss pre-post session (8%) was significant, and fatigue, measured as CMJ height loss, was strongly correlated to lactate (r = 0.97; p < 0.001) and ammonia (r = 0.92; p < 0.001) for all players. The relationships between the variables studied were determined by calculating the Pearson correlation coefficients. The metabolic stress developed during the effort can be estimated by controlling CMJ because of the high correlation between CMJ and blood lactate and ammonia concentrations. The high correlations found between mechanical (speed and CMJ height losses) and metabolic (lactate and ammonia) measures of fatigue highlight the utility and validity of using CMJ to monitor training load and quantify objectively neuromuscular fatigue during RSA.