In humans, plasma hyperosmolality delays the onset of sweating and cutaneous vasodilatation during heat stress. However, it remains unknown if hyperosmolality exerts this effect through a central (i.e. CNS) and/or peripheral (i.e. effector organ) modulation of thermoregulatory activity. We examined if intravenous infusion of hyperosmotic saline affects skin sympathetic nerve activity (SSNA) during whole-body passive heating in healthy humans. Furthermore, we examined if local intradermal infusion of hyperosmotic saline affects sweating and cutaneous vasodilatation during passive heating. Following intravenous infusion of either 0.9% (ISO) or 3.0% (HYPER) NaCl saline, 12 subjects were passively heated until core temperature increased by ∼0.6°C. During each condition, sweating and cutaneous vascular conductance were measured over two intradermal microdialysis probes, one perfused with ISO saline and the other with HYPER saline. Intravenous infusion of HYPER saline increased plasma osmolality (294±3 to 316±5 mOsmkg-1 H2O, P≤0.01), which remained greater than ISO throughout heating. Plasma hyperosmolality delayed the mean body temperature onset of sweating (+1.24±0.18vs. +1.60±0.18°C, P≤0.01) and cutaneous vasodilatation (+1.15±0.18vs. +1.53±0.22°C, P≤0.01), and attenuated the increase in SSNA during heating (+147±178vs. +427±281%, P≤0.01). Intradermal infusion of HYPER saline increased baseline cutaneous vascular conductance (P≤0.01), which did not increase further during the subsequent heating period (P=0.11). In contrast, intradermal infusion of HYPER saline did not affect sweating (P=0.99). These results provide direct evidence that plasma hyperosmolality exerts a central modulatory effect governing efferent thermoregulatory activity in humans.