Some sports participants are often reluctant to wear a mouthguard due to issues with comfort, breathing and communication. However, there is limited evidence that investigates the use of custom‐made mouthguards and variations in design as key factors to minimise these issues. Therefore, the aim of this study was to examine the effect of custom‐made mouthguard design on cardiopulmonary function, exercise performance and perception of comfort in rugby union players. Fourteen rugby players (aged 20.2 ± 1.19 years) were recruited to undertake a rugby‐specific exercise protocol on a treadmill over four conditions (no mouthguard and three custom‐made mouthguard designs). Cardiopulmonary responses were assessed using breath‐by‐breath analysis, in conjunction with blood lactate (BLa) and rating of perceived exertion (RPE). Maximum oxygen uptake (VO2max) was assessed before and after the study to identify any changes in players' level of performance. Participants scored each mouthguard in relation to preference, comfort, breathing, protection and retention. There were no differences in oxygen uptake (p = 0.785, = 0.021), ventilation (p = 0.952, = 0.007), respiratory exchange ratio (p = 0.564, = 0.039) and heart rate (p = 0.830, = 0.017), whilst participants performed with the selected custom‐made mouthguards. However, RPE was higher without a mouthguard than whilst using two of the mouthguards during the first 3 min of exercise, in combination with higher BLa accumulation (p ≤ 0.05). Although there was no statistical difference between mouthguard designs in the ratings around comfort, there was a preference towards mouthguards with reduced palatal coverage. Individual preference in design may improve compliance of wearing a mouthguard without affecting physiological parameters.