Resistance training has been shown to be a stressor factor on the autonomic nervous system, and these changes can be detected by heart rate variability (HRV) analysis. The aim of the present study was to evaluate the delayed effects of velocity loss-based resistance training strategies on heart rate variability (HRV), the sleep quality index (SQI) and delayed onset muscle soreness (DOMS). Fourteen men performed daily recordings. After a baseline period of 14 days of no training, they performed one session per week of resistance training focused on lower body exercise (squats) based on movement velocity. Three resistance training sessions composed of four sets up to 10%, 20% and 40% of velocity loss were performed each week of the study. Statistically significant changes (p < 0.05) after 24 hours of training were found in DOMS, and HRV variables, specifically in RR intervals (RR), root mean square of successive differences of RR intervals (RMSSD), and the percentage of successive RR intervals that differed by more than 50 ms (pNN50), between 40% of velocity movement loss and the rest of conditions. We can conclude that greater losses of execution velocity may result in greater internal load stimuli according to the autonomic modulation measured by HRV. RR, RMSSD and pNN50 seem to be the most sensitive indicators of HRV to fatigue produced by resistance training. This research opens the door to the study of HRV behavior related to resistance training. New research possibilities are raised by measuring the effect of guiding resistance training by means of HRV behavior.