The purpose of this study is to analyse the neuromuscular balance of the masticatory muscles and the influence on the athlete respiratory parameters induced by custom-made mouthguard. Twenty-six athletes (24 males and 2 females), of different disciplines, average age of 32.12 ± 12.05, were recruited. Each athlete received a custom-made mouthguard in the Ethylene-Vinyl-Acetate (EVA) thermoplastic material and using surface electromyography, the masseter muscles and the frontal beams of the temporal muscles were analysed, with and without mouthguard. The athletes were then subjected to a medical examination and cardiopulmonary test, in the condition with and without mouthguard. The use of mouthguard caused an improvement of all the electromyographic indexes analysed. In particular, GLOBAL INDEX (p = 0.0021), BAR (p = 0.0005), IMPACT (p = 0.0076) and ASIM (p = 0.0290) showed an enhancement statistically significant. For the cardiopulmonary test indexes as maximum oxygen uptake (VO 2 max), minute ventilation (VE), breathing reserve (BR), not statistically differences (p > 0.05) were reported. The custom-made mouthguard improved the neuromuscular balance of the masticatory muscles, symmetrizing the masseters and temporalis muscles work. It produced a better balancing distribution of occlusal loads both in the anterior-posterior direction and in the lateral direction and offered the possibility to produce more muscular work. Moreover, it did not constitute an impediment that can significantly disturb the athlete's breathing during physical activity.