Steroid hormone responses to three exercise modalities assessed by liquid chromatography tandem mass spectrometry in a randomized crossover trial

Abstract Exercise disrupts hormonal balance, driving physiological adaptations. However, inconsistent findings exist due to methodological limitations. To address this, this study simultaneously profiled 16 steroid hormones via LC-MS/MS in response to three distinct exercise modalities – moderate intensity continuous exercise (MICE), resistance exercise (RE), and High-intensity Intermittent Exercise (HIIE) – using a randomized crossover design. Ten healthy adult males completed HIIE (> 80% V̇O 2max ), MICE (< 65% V̇O 2max ) and whole-body RE (3 sets×10 reps at 70% 1RM with 90 s rest) in a randomized sequence. Blood samples were collected pre-, 10 min post-, and 2 h post-exercise, and analyzed for glucose, lactate, and steroid hormones. DHEA and androstenedione increased post-exercise following MICE, HIIE (both P < 0.01), and RE ( P < 0.05), but declined below rest by 2 h. Estrone increased 2 h post-RE vs. post-exercise ( P < 0.01), and 2 h post-HIIE vs. rest ( P < 0.01). Only HIIE elevated cortisol ( P < 0.001) and cortisone ( P < 0.01) post-exercise, while cortisol fell below rest at 2 h across all modalities ( P < 0.001). HIIE also raised corticosterone ( P < 0.001), exceeding post-MICE ( P < 0.01) which showed sub-resting corticosterone at 2 h post-exercise ( P < 0.01). All exercise modalities increased aldosterone ( P < 0.01), with greater responses in MICE and HIIE than RE. At 2 h, all protocols elevated the testosterone: cortisol ratio ( P < 0.05). RE and HIIE altered sex hormone ratios, decreasing testosterone:17b-estradiol and androgens: estrogens, and increasing estrogens: progesterone ( P < 0.05). These exploratory findings demonstrate mode-specific acute hormonal responses in young males. Future studies should explore circulating hormonal tissue interactions to clarify potential functional adaptations.