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This study investigated the effects of the menstrual cycle on prolonged exercise performance both in temperate (20°C, 45% relative humidity) and hot, humid (32°C, 60% relative humidity) conditions.For each environmental condition, 12 recreationally active females were tested during the early follicular (day 3–6) and midluteal (day 19–25) phases, verified by measurement of estradiol and progesterone. For all four tests, thermoregulatory, cardiorespiratory, and perceptual responses were measured during 60 min of exercise at 60% of maximal oxygen consumption followed by an incremental test to exhaustion.No differences in exercise performance between menstrual cycle phases were found during temperate conditions (n = 8) despite a higher resting and submaximal exercise core temperature (Tc) in the luteal phase. In hot, humid conditions (n = 8), however, prolonged exercise performance, as exercise time to fatigue, was significantly reduced during the luteal phase. This finding was not only accompanied by higher resting and submaximal exercise Tc but also a higher rate of increase in Tc during the luteal phase. Furthermore, submaximal exercise HR, minute ventilation, and RPE measures were higher during the luteal phase in hot, humid conditions. No significant differences were found over the menstrual cycle in heat loss responses (partitional calorimetry, sweat rate, upper arm sweat composition) and Tc at exhaustion.In temperate conditions, no changes in prolonged exercise performance were found over the menstrual cycle, whereas in hot, humid conditions, performance was decreased during the luteal phase. The combination of both exercise and heat stress with the elevated luteal phase Tc at the onset of exercise resulted in physiological and perceptual changes and a greater thermosensitivity, which may explain the decrease in performance.