This study investigated the acute fatigue pattern in neuromuscular activity after a simulated Boccia game and the effect of fatigue pattern on sport performance. Nine elite Boccia athletes were tested before, during, and after a simulated game. Maximum ball speed was captured with video, and the target hitting rate and rating of perceived exertion (RPE) score were collected and analyzed. Electromyography signals from the upper trapezius, anterior deltoid, posterior deltoid, biceps brachii, triceps brachii, and wrist extensor muscles were collected by surface electrode and were evaluated with mean power frequency (MPF). Only the upper trapezius muscle showed fatigue as demonstrated by a reduction of MPF of 8% (p = 0.027) when comparing the first and last throws in a simulated game. Subjective RPE score increased during the game (118%, p = 0.004), and sports performance in terms of maximum ball speed (-12%, p = 0.004) and target hitting rate (-25%, p = 0.004) also deteriorated. In conclusion, fatigue on the upper trapezius muscle was demonstrated in elite Boccia athletes following a prolonged Boccia game and may have affected Boccia performance. Preventative measures against upper trapezius muscle fatigue and endurance training for synergists of the upper trapezius muscle may be considered in future studies.