Exercise and hyperthermia can increase intestinal permeability and cause an influx of lipopolysaccharide (LPS) into the intravascular space. LPS initiates a robust pro-inflammatory cascade which can contribute to heat-related illnesses. Fluctuations in oestrogen throughout the menstrual cycle have been shown to modulate intestinal barrier function, with indirect evidence linking high oestrogen concentrations in the luteal phase with an improved intestinal barrier function. Improved barrier function may impact pro-inflammatory responses across the menstrual cycle in response to hyperthermia-mediated LPS influx. There is little research on whether pro-inflammatory responses to LPS and/or hyperthermia differ in eumenorrheic women compared to women who take an oral contraceptive, or whether responses fluctuate across monthly hormone/pill cycles. This study aimed to quantify monocyte release of pro-inflammatory cytokine tumour necrosis factor-alpha (TNFα) following LPS-stimulation with or without hyperthermia in whole blood obtained from oral contraceptive users and eumenorrheic women throughout the pill/menstrual cycle. Volunteers using oral contraceptives (OC, n=7) were tested during the pill-free phase (PF; day 6±1), and twice during the pill phase (P1; day 14±2, and P2; 21±2 including the pill-free week). Eumenorrheic volunteers (MC, n=7) were tested during the early follicular (EF; day 6±1), ovulation (OV; day 16±2), and mid-luteal (ML; 7±1 days’ following positive ovulation test) phases of the menstrual cycle. Participants rested for 20-minutes before a 20 mL venous blood sample was drawn into a syringe and transferred into sodium-citrate tubes. Whole blood (1 mL) was stimulated with 100μg.mL-1 of LPS and one aliquot was incubated at 37°C (body temperature), and another at 40°C (simulated hyperthermia) for 6 hours. Plasma was diluted 1:40 in 1% bovine serum albuminate in phosphate-buffered saline, and TNFα concentration determined via ELISA (R&D Duoset, within-plate coefficient of variation (CV) 4%, between-plate CV 14%). Absolute TNFα concentrations (ng.mL-1) were analysed by repeated-measures ANOVA, with temperature (37°C and 40°C) and time (OC: PF, P1, P2; MC, EF, OV, ML] phase) within-subject factors and group (OC and MC) a between-subject factor. Effect sizes are expressed at partial eta2 (ηp2). TNFα concentrations were 28% greater after incubation at 40°C compared to 37°C (main effect for temperature, p<0.001, ηp2=0.610). The temperature-mediated response was similar in both groups (temperature x group interaction, p=0.784, ηp2=0.006), regardless of menstrual cycle phase or pill phase (main effect of time, p=0.212, ηp2=0.121). The TNFα response was similar between P1 vs. PF (+4%), and P1 vs. P2 (+7%) in the OC group regardless of temperature. TNFα concentrations were increased between ML vs. EF (+39%) and between ML vs. OV (+55%) regardless of temperature, however, the time x group interaction did not meet the conventional threshold for statistical significance (p=0.068, ηp2=0.200). TNFα responses were similar between the two groups when stimulated with LPS with/or without hyperthermia. A larger pro-inflammatory response occurred during the ML phase of the MC  across both temperatures, despite evidence linking oestrogen with a reduction in pro-inflammatory responses. In conclusion, our data suggest that monocyte responses to immunogenic stimuli fluctuate throughout a hormone cycle in eumenorrheic women, but are more stable in those taking oral contraceptives.