The aim of this study was to investigate the effect of acute fatigue induced by a soccer simulation protocol on neuromuscular control indices (rate of force development [RFD] and reaction time [RT]) and dynamic balance (using the Y-Balance Test and Biodex Dynamic Stability Index) in professional football players. Twenty-four professional male players (mean age 23.4 ± 2.1 years, height 178.5 ± 5.2 cm, weight 72.3 ± 6.8 kg) were randomly assigned to an experimental group (with fatigue protocol) and a control group (without fatigue). The experimental group performed the SAFT protocol, consisting of 6 periods of 15-minute intermittent activity at 85-95% of maximum heart rate, while the control group remained at rest. Pre- and post-intervention, variables including RFD, RT, Y-Balance performance in three directions (anterior, posteromedial, and posterolateral), and Biodex Dynamic Stability Index were measured. Data analysis using paired t-tests and repeated-measures ANOVA showed that after acute fatigue, RFD significantly decreased (p = 0.01), RT increased (p = 0.02), Y-Balance performance weakened in all three directions (p < 0.05), and Biodex Dynamic Stability Index increased (p = 0.01), indicating reduced balance. No significant changes were observed in the control group. The findings suggest that acute fatigue can impair neuromuscular control and dynamic balance in professional players. These results emphasize the need for specialized training programs to enhance fatigue tolerance and implement post-fatigue recovery interventions to reduce injury risk and maintain performance.