ABSTRACT Introduction Regular physical exercise, or a more active lifestyle, are important to prevent cardiovascular diseases, reducing not only cardiovascular mortality but also promoting a reduction in the risk factors related to these diseases. Objective To assess the association between physical inactivity and heart rate variability (HRV) in adolescents. Methods This was a cross-sectional study of the Diagnostic Type with Level of Evidence II according to the Oxford table. One hundred and twenty-nine adolescents were evaluated, aged 15 and 17 years old, divided into four groups: male teenagers who were insufficiently active (IAM n = 28) or physically active (FAM n = 29), and female teenagers who were insufficiently active (IAF n = 42) or physically active (FAF n = 30). The level of physical activity was assessed by the IPAQ. The HRV was evaluated in the time and frequency domains. Results Reduced pulse interval variance was observed in the insufficiently active male or female groups (5089 ± 378 ms2 and 4335 ± 276 ms2 respectively) compared to the physically active groups (9106 ± 606 ms2 and 6182 ± 366 ms2 respectively). Moreover, the insufficiently active groups presented higher cardiac sympathetic/vagal balance values (0.81 ± 0.05 and 0.80 ± 0.05 respectively) compared to the physically active groups (0.63 ± 0.05 and 0.55 ± 0.05 respectively). Conclusion A physically active lifestyle was associated with better cardiovascular autonomic modulation in adolescents. Level of Evidence II; Diagnostic Studies - Investigating a Diagnostic Test.