Evolutionary perspectives of human behavior propose the existence of three emotion regulation systems (i.e., threat, drive and soothing systems). An unbalanced functioning of the systems represents greater risk for emotion dysregulation and psychopathology. In recent years, heart rate variability (HRV) has been reported as an accurate index of emotion regulation, and although adolescence is characterized by multiple neurophysiological, psychological and social changes, there is no study exploring the HRV patterns of each emotion regulation system in this developmental stage. In Study 1, a standardized procedure (SP) aiming to elicit the three different systems was developed and validated by experts (n = 14) and community adolescents (n = 31). In study 2, differences in HRV patterns across the three emotion regulation systems and across sex, were investigated in a sample of community adolescents (n = 155; 70 males), aged between 14 and 18 years old. Results showed that the threat and drive systems were associated with decreases in HRV, while the soothing system was associated with decreased heart rate. Sex differences were found for the activation of the threat system: while males maintained a decreasing trend in HRV indexes, from resting to recovery, females did not show a decrease in HRV during the activation of this system. Overall, physiological correlates of each specific emotion regulation system corroborate the theoretical assumptions. Moreover, a SP able to trigger each system independently while measuring physiological data is now available and can be used in future research.