Ca2+ is an important second messenger in eccrine sweating, with both internal and external sources identified in isolated rhesus monkey palm sweat glands1. It is unclear if these isolated gland studies can be translated to in vivo interstitial Ca2+ level modulation and if this in vivo modulation has the capacity to modulate sweat rate (SR) and skin blood flow (SkBF) in intact human forearm skin, which unlike palm skin, is vital to thermoregulation. We hypothesized that lowering interstitial Ca2+ levels or attenuating its influx would cause a rightward shift in the SR to acetylcholine (ACh) dose-response relation. 1) To identify that our methods had the capacity to modulate Ca2+ concentrations, osmolality was measured during Ca2+ or Ca2+ chelator (43 mM EDTA) perfusion through a microdialysis membrane into 2 ml distilled water with no Ca2+, 200 mM Ca2+, or EDTA. EDTA decreased perfusate and bath osmolality. To quantify Ca2+ in physiologic concentrations, EDTA in distilled water or lactated Ringers (2 mM Ca2+) was perfused through a bath containing the opposite solution. EDTA decreased perfusate and bath Ca2+ concentrations via microbore ion chromatography with conductivity detection. 2) 9 healthy human subjects received 6 ACh doses (1×10-5 – 1×100 M, 10-fold increments) with and without EDTA via forearm intradermal microdialysis. Capacitance hygrometry SR measures were completed directly over microdialysis membranes. SR to ACh dose-response modeling via nonlinear regression curve fitting (mean R2 0.97±0.05 with, 0.95±0.03 without EDTA) identified maximal responsiveness (Emax) and ED50 (ACh dose causing 50% Emax). EDTA right shifted the ED50 (1.5±0.1 -logM) compared to ACh alone (1.0±0.1 -logM; p=0.01). Emax was not different between groups (0.60±0.078 and 0.58±0.34 mg/cm2/min, respectively; p=0.85). 3) The EDTA microdialysis protocol was repeated in 5 subjects, measuring SkBF via laser-Doppler flowmetry to control for the potential of SkBF affecting SR independently. Neither Emax nor ED50 was significantly different between groups. 3) 10 healthy human subjects received the same 6 ACh doses with and without L-type Ca2+ channel blocker (1 mM verapamil) via forearm intradermal microdialysis. SR to ACh dose-response curve fitting (mean R2 0.96±0.01 with, 0.97±0.01 without verapamil) identified a right shifted ED50 (0.9±0.1 -logM) compared to ACh alone (1.6±0.2 -logM; p<0.01). Emax was not different between groups (0.45±0.08 and 0.35±0.06 mg/cm2/min, respectively; p=0.07). Thus, local in vivo Ca2+ chelation has the capacity to decrease interstitial Ca2+ concentration and to attenuate eccrine sweat gland cholinergic sensitivity. L-type Ca2+ channel antagonism also attenuates eccrine sweat gland cholinergic sensitivity. These data suggest that interstitial Ca2+ and its influx play a SkBF-independent role in human cholinergic-induced eccrine sweating that is important in thermoregulation.