The aim of this study was to investigate the association between cardiac parasympathetic activity and cardiorespiratory fitness, insulin, and hemodynamic profile in overweight and obese adolescent girls and boys (aged 12–16 years). Data were taken from the Multidisciplinary Obesity Treatment Program. Only post-intervention measurements are presented herein. Body composition, cardiorespiratory fitness, blood pressure, and metabolic profile (insulin and glucose profile) of adolescents were assessed. Cardiac parasympathetic activity was determined by resting heart rate variability, which was analyzed using a heart rate monitor. Greater parasympathetic cardiac activity was associated with higher levels of cardiorespiratory fitness in both girls and boys (0.375 ≤ r ≤ 0.900), while the sympathetic-vagal balance was negatively related to maximal oxygen uptake (VO2max) in girls (r = 0.478). An association between lower parasympathetic activity and insulin resistance was noted in girls (mean of R–R intervals [RRmean] and homeostasis model assessment insulin-resistance index [HOMA-IR]: r = −0.678), while greater systolic blood pressure (SBP) and lower parasympathetic activity were associated in both sexes (RRmean and SBP: r = −0.526; high frequency [HF (nu)] and SBP: r = −0.754). In conclusion, autonomic nervous system activity was associated with cardiorespiratory fitness, insulin resistance, and SBP in overweight and obese adolescents. The identification of these potential relationships assists with the establishment of future long-term exercise interventions that evaluate the improvements in parasympathetic nervous system activity, in addition to metabolic profile and cardiorespiratory fitness in overweight and obese adolescents.