<i>Background:</i> Toll-like receptors (TLRs) are pattern recognition receptors that play an important role as mediators of innate immunity. Human studies have shown changes in endometrial TLR expression during the menstrual cycle and during pregnancy. Our objective was to measure peripheral TLR activity over the course of the menstrual cycle. <i>Methods:</i> We recruited 11 healthy females, and using ELISA we measured sex hormone levels and IL-1β, IL-6, IL-8 and TNF-α following stimulation of whole blood with different TLR agonists during follicular, and early and late luteal phases of the menstrual cycle. <i>Results:</i> During the follicular phase, we observed lower levels of IL-6 and TNF-α following stimulation with the TLR2 agonist HKLM when compared with the early luteal phase; lower levels of IL-1β, IL-6 and TNF-α following stimulation with the TLR4 agonist LPS, and lower levels of IL-1β and TNF-α following stimulation with the TLR5 agonist flagellin. Decreased IL-6 levels in the late compared to the early luteal phase were also observed following stimulation with the TLR5 agonist flagellin. Compared with the follicular phase, the late luteal phase of the cycle resulted in decreased levels of IL-1β and TNF-α following stimulation with the TLR1/TLR2 agonist Pam3CSK and the TLR6/TLR2 agonist FSL1, as well as decreased levels of TNF-α following stimulation with the TLR8 agonist ssRNA40. There were no differences in cytokine release across the menstrual cycle following stimulation with the TLR3 agonist polyriboinosinic polyribocytidylic acid, or the TLR7 agonist Imiquimod. <i>Conclusion:</i> This study is the first to demonstrate that TLR responsivity in peripheral blood fluctuates throughout the menstrual cycle.