Beetroot juice (BJ) has been used as a sport supplement, improving performance in resistance training (RT). However, its effect on the modulation of the autonomic nervous system has not yet been widely studied. Therefore, the objective of this randomized double-blind crossover study was to assess the effect of acute BJ supplementation compared to placebo in blood pressure (BP), heart rate (HR), heart rate variability (HRV) and internal load during RT measure as Root Mean Square of the Successive Differences between adjacent RR intervals Slope (RMSSD and RMSSD-Slope, respectively). Eleven men performed an incremental RT test (three sets at 60%, 70% and 80% of their repetition maximum) composed by back squat and bench press with. HR, HRV and RMSSD-Slope were measured during and post exercise. As the main results, RMSSD during exercise decrease in the BJ group compared to placebo (p = 0.023; ES = 0.999), there were no differences in RMSSD post-exercise, and there were differences in RMSSD-Slope between groups in favor of the BJ group (p = 0.025; ES = 1.104) with a lower internal load. In conclusion, BJ supplementation seems to be a valuable tool for the reduction in the internal load of exercise during RT measured as RMSSD-Slope while enhancing performance.