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Background: Cystic fibrosis (CF) affects the autonomic nervous system (ANS) and exercise in healthy children modulates the interaction between sympathetic and parasympathetic activity. This study aimed to evaluate the effects of a short-term resistance exercise program on heart rate variability (HRV) in children and adolescents with CF.Methods: A randomized controlled trial was carried out in children diagnosed with CF aged 6–18 years. Individuals were divided into two groups: control (CON) and resistance-training (EX). Individuals in the EX group completed an individualized guided resistance program (5-RM—60–80%) for 8 weeks (3 sessions of 60 min/week). Upper and lower limbs exercises (seated bench press, seated lateral row, and leg press) were used. HRV was measured using a Suunto watch with subjects in lying position.Results: Nineteen subjects (13 boys) were included (CON = 11; and EX = 8). Mean age was 12.2 ± 3.3, FEV1 (forced expiratory volume in the first second) z-score was 1.72 ± 1.54 and peak oxygen consumption (VO2peak) 42.7 ± 7.4 mL.Kg–1.min–1. Exercise induced significant changes in the frequency-domain variables, including a decrease in LF power (p = 0.001, d = 0.98) and LF/HF ratio (p = 0.020, d = 0.92), and an increase in HF power (p = 0.001, d = −0.97), compared to the CON group. No significant changes were found for time-domain variables, although increases with a moderate effect size were seen for SDNN (p = 0.152, d = −0.41) and RMSSD (p = 0.059, d = −0.49) compared to the CON group.Conclusion: A short-term resistance exercise-training program was able to modulate HRV in children and adolescents with CF presenting mild to moderate lung function impairment and good physical condition.Clinical Trial Registration:www.ClinicalTrials.gov, identifier NCT04293926.