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

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.