Oxidative stress has been shown to have a negative effect on the autonomic nervous system (ANS) and on ANS modulation of heart rate. Mitochondrial ATP production is the main source of reactive oxygen species and hence the regulation of ROS becomes an important issue in maintaining optimal ANS functionality. Humanin (HN), a mitochondrial-derived peptide, plays an important role in lowering oxidative stress (OS). Sympathovagal balance was assessed in 124 healthy participants through heart rate variability (HRV) analysis and compared across changes in HN concentrations divided into quintiles. Significant differences included various frequency domain and nonlinear HRV parameters, particularly between first and fourth HN quintiles. The results revealed non- monotonic relationships between measures of HRV and HN concentration. A mitohormetic type of relationship was observed with HRV features increasing and then decreasing with increasing HN concentration These results are consistent with previous findings of the importance of HN levels in regulating OS and extend these by revealing a concomitant effect on the modulation of cardiac rhythm by the ANS.