Chronic pain is a pervasive health problem and is associated with tremendous socioeconomic costs. However, current pain treatments are often ineffective due, in part, to the multi-factorial nature of pain. Mild hypohydration was shown to increase experimental pain sensitivity in men, but whether this also occurs in women has not been examined. Fluctuations in ovarian hormones (i.e., 17ß-oestradiol and progesterone) throughout the menstrual cycle may influence a woman's pain sensitivity, as well as hydration levels, suggesting possible interactions between hypohydration and menstrual phase on pain. We investigated the effects of mild hypohydration (HYPO, 24 hr of fluid restriction) on ischaemic pain sensitivity in 14 eumenorrheic women during the early follicular (EF) and mid-luteal (ML) phases of their menstrual cycle. We also examined whether acute water ingestion could reverse the negative effects of hypohydration. Elevated serum osmolality, plasma copeptin, and urine specific gravity indicated mild hypohydration. Compared to euhydration, HYPO reduced pain tolerance (by 34 ± 46 s; P = 0.02, ηp2 = 0.37) and increased ratings of pain intensity (by 0.7 ± 0.7 cm; P = 0.004; ηp2 = 0.55) and unpleasantness (by 0.7 ± 0.9 cm; P = 0.02; ηp2 = 0.40); these results were not influenced by menstrual phase. Water ingestion reduced thirst perception (Visual Analogue Scale, by 2.3 ± 0.9 cm; P < 0.001, ηp2 = 0.88) but did not reduce pain sensitivity. Therefore, hypohydration increases pain sensitivity in women with no influence of menstrual phase.