The aim of this study is to analyse the adaptations of the autonomic nervous system after a musculoskeletal injury, obtained by measuring heart rate variability in athletes. It was hypothesized that there is an alteration in heart rate variability after a musculoskeletal injury. Cohort study. 15 semi-professional soccer players from three football teams, aged between 21 and 33 (mean age: 29.4 ± 3.31 years), with a recent musculoskeletal injury. Heart rate variability was collected using the Polar m200 and the chest strap H10 in two moments: within 72 h after the injury and between 5 and 7 days after full return-to-play. Results show differences between T1 and T2 (p ≤ 0.05) in low-frequency power (n.u.) (p = 0.001) and high-frequency power (n.u.) (p = 0.001), in low-frequency/high-frequency ratio (p = 0.001) and in high-frequency power (ms2) (p = 0.017) measures. No statistical differences were found in low-frequency power (ms2) (p = 0.233). The low frequency power (n.u.) was significantly lower after injury compared with LF power (n.u.) values after full return-to-play. In high-frequency power there was a significant difference between both moments with high values after injury. The use of heart rate variability therefore seems to be promising to detect an imbalance in the autonomic nervous system and help clinical departments to identify a possible non-traumatic musculoskeletal injury. Further research should be performed considering a wide range of musculoskeletal injuries and to establish baseline values of the athletes.