Purpose <p>Impulsivity, a personality construct linked to various psychiatric disorders, is partially regulated by serotonergic neurotransmission. While genetic polymorphisms affecting serotonin transporter (5-HTT) transcription have been widely studied, the role of epigenetic modifications, particularly DNA methylation, remains unclear. Building on earlier analyses from the present cohort that demonstrated that eight weeks of physical exercise reduces impulsivity, this study investigated whether these behavioural changes are accompanied by alterations in 5-HTT expression together with 5-HTT and MAO-A promoter methylation in buffy coat (BC) cells.</p> Registry <p> drks.de, TRN: DRKS00016589, Registration date: 6 February 2019.</p> Methods <p>Participants (<i>n</i> = 45) were randomly assigned to either high-intensity interval training (HIIT) or stretching (active control). Each group completed three training sessions per week for eight weeks, with pre- and post-intervention assessments of impulsivity, 5-HTT gene expression, and 5-HTT and MAO-A promoter methylation.</p> Results <p>Results indicate a significant increase in 5-HTT expression in the HIIT group compared to controls (<i>p</i> = .008, <i>η</i>_<i>p</i>^<i>²</i> = 0.156; <i>d</i> = 0.782), but no corresponding changes in promoter DNA methylation. Moreover, changes in 5-HTT expression did not correlate with changes in impulsivity. Specific 5-HTT and MAO-A promoter methylation changes were weakly associated with certain impulsivity factors.</p> Conclusion <p>These findings suggest that exercise influences serotonergic function by increasing 5-HTT expression independently of promoter DNA methylation changes. Further research is needed to determine whether these changes are also present at central nervous system level or if this upregulation is primarily a result of long-term anti-inflammatory effects triggered by physical exercise.</p>