Tobacco smoking using a waterpipe (narghile, hookah, shisha) has become a global epidemic. Unlike cigarette smoking, little is known about the health effects of waterpipe use. One acute effect of cigarette smoke inhalation is dysfunction in autonomic regulation of the cardiac cycle, as indicated by reduction in heart rate variability (HRV). Reduced HRV is implicated in adverse cardiovascular health outcomes, and is associated with inhalation exposure-induced oxidative stress. Using a 32 participant cross-over study design, we investigated toxicant exposure and effects of waterpipe smoking on heart rate variability when, under controlled conditions, participants smoked a tobacco-based and a tobacco-free waterpipe product promoted as an alternative for “health-conscious” users. Outcome measures included HRV, exhaled breath carbon monoxide (CO), plasma nicotine, and puff topography, which were measured at times prior to, during, and after smoking. We found that waterpipe use acutely decreased HRV (p<0.01 for all measures), independent of product smoked. Plasma nicotine, blood pressure, and heart rate increased only with the tobacco-based product (p<0.01), while CO increased with both products (p<0.01). More smoke was inhaled during tobacco-free product use, potentially reflecting attempted regulation of nicotine intake. The data thus indicate that waterpipe smoking acutely compromises cardiac autonomic function, and does so through exposure to smoke constituents other than nicotine.