Objective <p>Chronic sleep deprivation (SD) and caffeine consumption individually impair male reproductive function; however, the combined impact of simultaneous exposure on reproductive neuroendocrinology remains largely unexplored. This study investigated the effects of concomitant exposure to a novel Unpredictable Chronic Sleep Deprivation (CUSD) paradigm and high-dose caffeine on hypothalamic monoaminergic neurotransmission, seminal indices, and hypothalamic-pituitary-adrenal/gonadal (HPA/HPG) axis activity in adult male Long-Evans rats.</p> Results <p>Twenty male Long-Evans rats were exposed to either CUSD, caffeine, or a combination of CUSD and caffeine. Combined CUSD and caffeine exposure significantly increased intromission latency and reduced intromission number, mounting latency, and mounting frequency compared to controls. Seminal parameters, including sperm motility, morphology, viability, and count, were markedly impaired in the CUSD and CUSD+caffeine groups. Serum GnRH, testosterone, LH, and FSH were significantly reduced, while corticosterone was significantly elevated across treatment groups, with the most pronounced effects in the CUSD+caffeine group. Hypothalamic serotonin and dopamine were significantly depleted in the CUSD and CUSD+caffeine groups. Nitrergic stress, evidenced by elevated iNOS and NO levels, was observed in hypothalamic and testicular tissues. These findings suggest that simultaneous exposure to CUSD and high-dose caffeine synergistically impairs male sexual performance and fecundity by dysregulating monoaminergic neurotransmission and HPA/HPG axis activity.</p> Graphical Abstract <p></p>