Background: Low participation rates are one of the most serious disadvantages of Web-based studies. It is necessary to develop effective strategies to improve participation rates to obtain sufficient data. Objective: The objective of this trial was to investigate the effect of emphasizing the incentive in the subject line of the invitation email and the day of the week of sending the invitation email on the participation rate in a Web-based trial. Methods: We conducted a 2�2 factorial design randomized controlled trial. We contacted 2000 primary care physicians from members of the Japan Primary Care Association in January 2017 and randomly allocated them to 1 of 4 combinations of 2 subject lines (presence or absence of an emphasis on a lottery for an Amazon gift card worth 3000 yen or approximately US $30) and 2 delivery days (sending the invitation email on Tuesday or Friday). The primary outcome was the response rate defined as the number of participants answering the first page of the questionnaire divided by the number of invitation emails delivered. All outcomes were collected between January 17, 2017, and February 8, 2017. Results: We analyzed data from 1943 out of 2000 participants after excluding those whose email addresses were invalid. The overall response rate was 6.3% (123/1943). There was no significant difference in the response rates between the 2 groups regarding incentive in the subject line: the risk ratio was 1.12 (95% CI 0.80 to 1.58) and the risk difference was 0.7% (95% CI -1.5% to 2.9%). Similarly, there was no significant difference in the response rates between the 2 groups regarding sending the email on Tuesday or Friday: the risk ratio was 0.98 (95% CI 0.70 to 1.38) and the risk difference was -0.1% (95% CI -2.3% to 2.1%). Conclusions: Neither emphasizing the incentive in the subject line of the invitation email nor varying the day of the week the invitation email was sent led to a meaningful increase in response rates in a Web-based trial with primary care physicians.