Unaccustomed strenuous physical exertion in hot environments can result in heat stroke and acute kidney injury (AKI). Both exercise-induced muscle damage and AKI are associated with the release of interleukin-6 but whether muscle damage causes AKI in the heat is unknown. We hypothesized that muscle damaging exercise, prior to exercise in the heat would increase kidney stress. Ten healthy, euhydrated males underwent a randomized, crossover trial involving both a 60 minute downhill muscle-damaging run (EIMD), and an exercise intensity-matched non-muscle damaging flat run (CON), in random order separated by two weeks. Both treatments were followed by heat stress elicited by a 40 minute run at 33oC. Urine and blood were sampled at baseline, after treatment, and after running in the heat. By design, EIMD induced higher plasma creatine kinase and interleukin-6 than CON. EIMD elevated kidney injury biomarkers (e.g. urinary NGAL after running in the heat: EIMD-CON, mean diff [95%CI]: 12 [5, 19] ng/ml) and reduced kidney function (e.g. plasma creatinine after running in the heat: EIMD-CON, mean diff [95%CI]: 0.2 [0.1, 0.3] mg/dl). Plasma interleukin-6 was positively correlated with plasma NGAL (r = 0.9, P = 0.001). Moreover, following EIMD, 5 of 10 participants met AKIN criteria for AKI. Thus, for the first time we demonstrate that muscle damaging exercise prior to running in the heat results in a greater inflammatory state and kidney stress compared to non-muscle damaging exercise. Muscle damage should therefore be considered a risk factor for AKI when performing exercise in hot environments.