introduction: with rising ambient temperatures globally, heat-vulnerable populations are at greater risk of negative health consequences. of increasing concern is the potential impact on human milk production and potential subsequent infant growth and development in our warming climate. while evidence in animals suggests a negative impact on milk production during periods of heat stress, whether acute heat stress modulates human milk production in postpartum mothers remains largely unexplored. method: ten lactating postpartum mothers (32 ± 3 years, 24.6 ± 3.5 kg/m², 33 ± 11 weeks postpartum) completed a 48-h experimental protocol whereby 24-h lactation performance was assessed before and after a one-hour passive heat stress intervention using a water-perfused suit at 49°c. throughout the 48-h intervention, mothers recorded their lactation performance as human milk production using a baby scale accurate to +/- 2 grams, fluid intake, and wore a fitbit charge5 to monitor activity levels. heart rate, blood pressure, and oral temperature were recorded before the intervention, immediately before and after the acute passive heating, and at the end of the intervention. whole-body sweat losses were measured during the acute passive heat stress. results: passive heat stress resulted in 343ml± 190g of whole-body sweat losses, and increased heart rate (+12±5 bpm, p<0.0001) and oral temperature (+0.4 ± 0.2°c, p=0.021). 24-h human milk volume was not different after (335 ± 156ml) compared to before passive heating (333 ± 118 ml) and did not alter milk production (p=0.921). additionally, participants consumed more fluid during the 24h after (1565 ± 70ml) compared to before (1325± 190ml, p=0.005). conclusion: acute whole-body heat stress does not significantly affect the 24-hour human milk production in breastfeeding mothers likely due to increase fluid intake.