Latent virus reactivation and diurnal salivary cortisol and dehydroepiandrosterone were measured prospectively in 17 astronauts (16 male and 1 female) before, during, and after short-duration (12-16 days) Space Shuttle missions. Blood, urine, and saliva samples were collected during each of these phases. Antiviral antibodies and viral load (DNA) were measured for Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and cytomegalovirus (CMV). Three astronauts did not shed any virus in any of their samples collected before, during, or after flight. EBV was shed in the saliva in all of the remaining 14 astronauts during all 3 phases of flight. Seven of the 14 EBV-shedding subjects also shed VZV during and after the flight in their saliva samples, and 8 of 14 EBV-shedders also shed CMV in their urine samples before, during, and after flight. In 6 of 14 crewmembers, all 3 target viruses were shed during one or more flight phases. Both EBV and VZV DNA copies were elevated during the flight phase relative to preflight or post-flight levels. EBV DNA in peripheral blood was increased preflight relative to post-flight. Eighteen healthy controls were also included in the study. Approximately 2-5% of controls shed EBV while none shed VZV or CMV. Salivary cortisol measured preflight and during flight were elevated relative to post-flight. In contrast DHEA decreased during the flight phase relative to both preflight and post-flight. As a consequence, the molar ratio of the area under the diurnal curve of cortisol to DHEA with respect to ground (AUCg) increased significantly during flight. This ratio was unrelated to viral shedding. In summary, three herpes viruses can reactivate individually or in combination during spaceflight.