As the impact of high intensity interval training (HIIT) on systemic hormones in aging men is unstudied to date, we investigated whether total testosterone (TT), sex hormone binding globulin (SHBG), free testosterone (free-T), and cortisol (all in serum) were altered following HIIT in a cohort of 22 lifelong sedentary (62 ± 2 years) older men. As HIIT requires preconditioning exercise in sedentary cohorts, participants were tested at three phases, each separated by six weeks' training; baseline (phase A), following conditioning exercise (phase B), and post-HIIT (phase C). Each measurement phase used identical methods. TT was significantly increased following HIIT (~17%; P<0.001) with most increase occurring during preconditioning (~10%; P=0.007). Free-T was unaffected by conditioning exercise (P=0.102) but was significantly higher following HIIT compared to baseline (~4.5%; P=0.023). Cortisol remained unchanged from A to C (P=0.138). The present data indicate a combination of preconditioning and HIIT increases TT and SHBG in sedentary older males, with the HIIT stimulus accounting for a small but statistically significant increase in free-T. Further study is required to determine the biological importance of small improvements in free-T in aging men.