Purpose: Microcirculatory dysfunction plays a key role in the development of sepsis during which core temperature is often disturbed. Skin microvascular assessment using laser techniques has been suggested to evaluate microvascular dysfunction during sepsis, but skin microcirculation is also a major effector of human thermoregulation. Therefore we aimed to study the effect of skin temperature on endothelial- and non-endothelial microvascular responses. Methods: Fifteen healthy participants were studied at different randomized ambient temperatures leading to low (28.0 +/− 2.0 °C), intermediate (31.6 +/− 2.1 °C), and high (34.1 +/− 1.3 °C) skin temperatures. We measured skin blood flow using laser speckle contrast imaging on the forearm in response to vasodilator microvascular tests: acetylcholine (ACh) iontophoresis, sodium nitroprussiate (SNP) iontophoresis, and post-occlusive reactive hyperemia (PORH). The results are expressed as absolute (laser speckle perfusion units, LSPU) or normalized values (cutaneous vascular conductance, CVC in LSPU/mmHg and multiple of baseline). Results: Maximal vasodilation induced by these tests is modified by skin temperature. A low skin temperature induced a significant lower vasodilation for all microvascular tests when results are expressed either in absolute values or in CVC. For example, ACh peak was 57.6 +/− 19.6 LSPU, 66.8 +/− 22.2 LSPU and 88.5 +/− 13.0 LSPU for low, intermediate and high skin temperature respectively (p < 0.05). When results are expressed in multiple of baseline, statistical difference disappeared. Conclusions: These results suggest that skin temperature has to be well controlled when performing microvascular assessments in order to avoid any bias. The effect of skin temperature can be corrected by expressing the results in multiple of baseline.