Social media is rapidly becoming the primary source to disseminate invitations to the public to consider taking part in research studies. There is, however, little information on how the contents of the advertisement can be communicated to facilitate engagement and subsequently promote intentions to participate in research. This paper describes an experimental study that tested different behavioral messages for recruiting study participants for a real-life observational case-control study. We included 1060 women in a web-based experiment and randomized them to 1 of 3 experimental conditions: standard advertisement (n=360), patient endorsement advertisement (n=345), and social norms advertisement (n=355). After seeing 1 of the 3 advertisements, participants were asked to state (1) their intention to take part in the advertised case-control study, (2) the ease of understanding the message and study aims, and (3) their willingness to be redirected to the website of the case-control study after completing the survey. Individuals were further asked to suggest ways to improve the messages. Intentions were compared between groups using ordinal logistic regression, reported in percentages, adjusted odds ratio (aOR), and 95% CIs. Those who were in the patient endorsement and social norms-based advertisement groups had significantly lower intentions to take part in the advertised study compared with those in the standard advertisement group (aOR 0.73, 95% CI 0.55-0.97; P=.03 and aOR 0.69, 95% CI 0.52-0.92; P=.009, respectively). The patient endorsement advertisement was perceived to be more difficult to understand (aOR 0.65, 95% CI 0.48-0.87; P=.004) and to communicate the study aims less clearly (aOR 0.72, 95% CI 0.55-0.95; P=.01). While the patient endorsement advertisement had no impact on intention to visit the main study website, the social norms advertisement decreased willingness compared with the standard advertisement group (157/355, 44.2% vs 191/360, 53.1%; aOR 0.74, 95% CI 0.54-0.99; P=.02). The majority of participants (395/609, 64.8%) stated that the messages did not require changes, but some preferred clearer (75/609, 12.3%) and shorter (59/609, 9.7%) messages. The results of this study indicate that adding normative behavioral messages to simulated tweets decreased participant intention to take part in our web-based case-control study, as this made the tweet harder to understand. This suggests that simple messages should be used for participant recruitment through Twitter (subsequently rebranded X). [Abstract copyright: ©Sandro T Stoffel, Jing Hui Law, Robert Kerrison, Hannah R Brewer, James M Flanagan, Yasemin Hirst. Originally published in JMIR Formative Research (https://formative.jmir.org), 05.02.2024.]