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Introduction E-cigarette use is disparately high among sexual minoritized populations. As e-cigarette advertising may influence product appeal, this study tested sexual orientation- and gender-based differences in response to e-cigarette advertisement exposure on advertisement perceptions and product appeal. Methods We recruited 497 adults (mean age=31.9 years, 45.1% women, 54.3% heterosexual, 71.2% Non-Hispanic White) living in the United States via the crowdsourcing platform Prolific. Participants viewed two randomly selected e-cigarette advertisements (from n=173 advertisements). Post-exposure, participants rated the perceived advertisement effectiveness, relevance, and product use intention. Associations between sexual orientation and outcomes were estimated using multivariable linear mixed-effects models. We tested interaction effects between sexual orientation, gender, and advertisement feature (e.g. presence of humans, flavors, and product packaging), and ran Tukey post hoc tests for pairwise comparisons. Results Post-exposure, heterosexual women, sexual minoritized men, and sexual minoritized women (reference group: heterosexual men) rated perceived advertisement effectiveness and relevance lower after viewing advertisements featuring flavors (vs no flavors; all p |