Background: The acute cardiovascular effects of electronic nicotine delivery devices (ENDs) have not been well-characterized. We hypothesized differences in acute cardiovascular and autonomic function responses to use of ENDS by chronic ENDs users (“vapers”), use of combustible cigarettes by chronic “smokers,” and no product use among non-smoking/non-vaping controls. Methods: CLUES (HL1393301) was a cross-sectional study of 395 individuals: 164 exclusive vapers (exhaled carbon monoxide [CO] <5 ppm, positive urine NicCheck I test), 117 exclusive smokers (CO >5 ppm, positive urine NicCheck), and 114 non-vaping/non-smoking controls (CO <5 ppm, negative urine NicCheck). We measured systolic and diastolic blood pressure (BP), heart rate (HR), brachial artery diameter, and time domain HR variability (root mean square differences in successive normal intervals [RMSSD]; % adjacent normal intervals >50 ms [PNN50]) before and 15’ after a product use challenge. Linear mixed models were created to predict outcome measures from group, time, and group*time with age, sex, and race as covariates, followed by group contrasts via post-hoc t-tests with Bonferroni-corrected p values. Results: Vapers were a mean (SD) 27.4 (10.6) years old (39% female, 86% white) and had vaped for 4.1 (2.7) years. Smokers were 42.8 (13.8) years old (44% female, 56% white) and had smoked for 23.0 (13.0) years. Controls were 30.8 (11.9) years old (50% female, 69% white). Model-adjusted mean (95% CIs) outcomes and p values for between group differences in challenge responses are in the Figure. Compared to controls, vapers had greater increases in systolic BP, diastolic BP, and HR (all p < 0.002) and greater reductions in brachial artery diameter, RMSSD, and PNN50 (all p < 0.003) than controls, with values similar to smokers. Conclusion: Chronic vapers showed acute increases in BP and HR, vasoconstriction, and reductions in time domain HR variability after using ENDS, similar to cigarette use by smokers.