Burn injuries occur over 1 million times annually and result in 40,000-70,000 hospitalizations in the United States. Burns >20% of total body surface area (%TBSA) often necessitate skin grafting. Grafted skin is unable to efficiently dissipate heat due to destruction of neural synapses, eccrine glands, and microvascular dysfunction. At donor sites, in which the sweat gland duct and coil are separated upon tissue translocation, complete regeneration may not occur. Therefore, we aimed to (1) measure sweat gland density, sweat rate, skin blood flow, and skin temperature at donor and adjacent skin sites in burn survivors, and (2) assess whether the differences in skin blood flow and gland function at each site induced by acetylcholine analogue depend on core temperature. METHODS Three participants (33.0 ± 11.5 years) received transdermal iontophoresis of pilocarpine at donor and adjacent sites prior and subsequent to an active heat stress protocol of 30 min walking at 40°C and 50% humidity. RESULTS No differences in sweat rate across pilocarpine and pilocarpine + heat stress conditions were present at the donor or control sites (p > 0.9999). Skin blood flow at the donor site following pilocarpine + heat stress (mean = 13.87, CI95 -16.64, 44.37) was higher than that at baseline (mean = 0.900, CI95 .1548, 1.645) (p = .0343), reflecting normal vasodilatory action. However, this effect was not core temperature-dependent. Sweat gland density was not different between the donor or control sites (Friedman’s Q = 0.667, p = 0.9444 and Friedman’s Q = 2.364, p = 0.3889). Sweat gland output was not different between heat stress or pilocarpine conditions at the donor or control sites (p = 0.7500 and p > 0.9999). Skin temperature was similarly elevated at the donor and control sites following heat stress (Friedman’s Q = 7.400, p = .0330 and Friedman’s Q = 8.200, p = .0174). CONCLUSIONS We found no evidence of low sweat gland density or thermoregulatory dysfunction at the donor skin site relative to adjacent unaffected skin. The donor site responded similarly when challenged with pilocarpine iontophoresis and heat stress.