Calcium is an important second messenger in eccrine sweating, with both internal and external sources being identified in vitro. It is unclear whether in vivo modulation of extracellular Ca²⁺ levels or influx has the capacity to modulate sweat rate in non‐glabrous human skin. To test the hypothesis that lowering interstitial Ca²⁺ levels would decrease the sensitivity of the ACh to sweat rate (via capacitance hygrometry) dose–response relationship, nine healthy subjects received six ACh doses (1 × 10⁻⁵ to 1 × 10⁰ m in 10‐fold increments) with and without a Ca²⁺ chelator (12.5 mg ml⁻¹ EDTA) via forearm intradermal microdialysis (protocol 1). To test the hypothesis that attenuating Ca²⁺ influx via L‐type Ca²⁺ channels would also decrease the sensitivity of the ACh to sweat rate dose–response relationship, 10 healthy subjects received similar ACh doses with and without a phenylalkylamine Ca²⁺ channel blocker (1 mm verapamil; protocol 2). Non‐linear regression curve fitting identified a right‐shifted ED₅₀ in EDTA‐treated sites compared with ACh alone (−1.0 ± 0.1 and −1.5 ± 0.1 logm, respectively; P < 0.05), but unchanged maximal sweat rate (0.60 ± 0.07 and 0.58 ± 0.11 mg cm⁻² min⁻¹, respectively; P > 0.05) in protocol 1. Protocol 2 also resulted in a right‐shifted ED₅₀ (verapamil, −0.9 ± 0.1 logm; ACh alone, −1.6 ± 0.2 logm; P < 0.05), with unchanged maximal sweat rate (verapamil, 0.45 ± 0.08 mg cm⁻² min⁻¹; ACh alone, 0.35 ± 0.06 mg cm⁻² min⁻¹; P > 0.05). Thus, local in vivo Ca²⁺ chelation and L‐type Ca²⁺ channel antagonism have the capacity to attenuate in vivo cholinergic sensitivity of eccrine sweat glands. These data suggest that interstitial Ca²⁺ and its influx via Ca²⁺ channels play a functional role in eccrine sweating in intact non‐glabrous human skin.