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Evidence indicates that master athletes have higher concentration of Sirtuin 1 (Sirt1), lower body fat (BF), and greater activity of the hypothalamic–pituitary–gonadal axis in comparison to untrained peers. However, no published data have demonstrated possible mediation effect of Sirt1 in the interaction of BF and testosterone in this population. Therefore, this study compared and verified possible associations between Sirt1, BF, fat mass index (FMI), testosterone, luteinizing hormone (LH), and testosterone/luteinizing hormone (T/LH) ratio in middle-aged master athletes (n = 54; 51.22 ± 7.76 years) and control middle-aged peers (n = 21; 47.76 ± 8.47 years). Venous blood was collected for testosterone, LH, and Sirt1. BF was assessed through skinfold protocol. Although LH concentration did not differ between groups, master athletes presented higher concentration of Sirt1, testosterone, and T/LH ratio, and lower BF and FMI in relation to age-matched nonathletes. Moreover, Sirt1 correlated positively with testosterone and T/LH ratio, negatively with BF, and was not significantly correlated with LH (mediation analysis revealed the effect of BF on testosterone is mediated by Sirt1 and vice versa; R2 =.1776; p =.032). In conclusion, master athletes have higher testosterone, T/LH ratio, and Sirt1, and lower BF and FMI in relation to untrained peers. Furthermore, Sirt1 was negatively associated with BF and positively associated with testosterone and T/LH ratio. These findings suggest that increased circulating Sirt1, possibly due to the master athlete's training regimens and lifestyle, exhibits a potential mediation effect on the interaction between endocrine function and body composition. [ABSTRACT FROM AUTHOR]