Identifying strategies to enhance vaccine response supports public health efforts, particularly in settings where individuals face elevated exposure risks, such as during military deployment or in other high-demand contexts. Accumulating evidence suggests that longer sleep duration may enhance vaccine response, but the role of sleep quality and timing remain less well understood. Additionally, although the circadian system interacts with the immune system, it is unclear whether improved vaccination response with morning administration is driven by internal biological timing. In this study, we examined both objective and subjective measures of sleep quantity, quality, and timing before, immediately after, and in the weeks following influenza vaccination to determine how sleep and vaccine response interact over time in individuals with and without 2 weeks of randomly assigned, exogenous melatonin post-vaccination. Sleep patterns were minimally different between treatment groups; however, the melatonin group reported worse sleep quality pre-vaccination and shorter sleep latency on the first night post-vaccination as compared to controls. After adjusting for sex, age, group, and baseline levels, better pre-vaccination sleep quality was associated with stronger immune responses, including higher hemagglutination inhibition (HAI) titers for the Victoria lineage and elevated fluorospot interferon gamma (IFN-Ξ³), granzyme B (GzB), and IFN-Ξ³+GzB responses (all p < 0.05). In contrast, associations between post-vaccination sleep and immune outcomes were more variable and marker-dependent. Chronotype was also associated with immune function, with earlier chronotypes exhibiting more robust responses across multiple measures. Mediation analysis also confirmed that our previously-reported increase in fold-rise IFN-Ξ³+GzB in the melatonin group was not attributable to sleep, either directly or indirectly. These findings suggest that sleep exerts complex, time-dependent effects on vaccine response, independent of melatonin supplementation. Moreover, chronotype and time of vaccination should be considered in future immunization studies. Ultimately, improving sleep quality prior to vaccination and administering melatonin post-vaccination may improve vaccine immune response.
