Background: In the Netherlands, human papillomavirus (HPV) vaccination uptake remains low. To improve informed decision making (IDM) and HPV vaccination acceptability, we systematically developed an interactive, web-based tailored intervention to which mothers of Dutch girls were invited to participate. Objective: The aim of this study was to provide insight into the intervention’s working mechanisms by evaluating (1) program use, (2) program acceptability, and (3) the relationship of program use with program acceptability and intervention effects (ie, dose-response relationship). Methods: Only mothers from the intervention arm of a randomized controlled trial that assessed the effectiveness of the web-based, tailored intervention were included in this study. They were invited to visit the website of the web-based intervention between baseline (January 2015, just before access to the intervention) and follow up (March 2015, prior to the first HPV vaccination). Indicators for program use were time of website use (ie, duration of intervention exposure) and completeness (ie, proportion of all available web pages visited). HPV vaccination uptake registered by Praeventis was used as the primary outcome. Secondary outcomes were IDM, decisional conflict, and social-psychological determinants of HPV vaccination uptake. Results: Among the 3995 invited mothers, 2509 (62.80%) logged on to the website, 2239 of whom (89.24%) visited at least one page of the intervention components. On average, mothers spent 21.39 minutes (SD 12.41) on the website and completed 50.04% (SD 26.18%) of the website components. Participants rated the website 7.64 (SD 1.39) on a 10-point scale. Program acceptability was significantly associated with completeness (β=4.36, P<.001), but not with time of website use (β=–.07, P=.77). Intention-to-treat analysis (N=3995) showed a significant positive effect of completeness on all outcome measures (all P<.003; Bonferroni-corrected α=.05/15 factors), including on HPV vaccination uptake. Time of website use had a significant positive effect on all outcomes (all P<.003), except for uptake (P=.20), risk perception when not vaccinated (P=.14), subjective norms (P=.03), and habit (P=.01). Conclusions: Program use and acceptability of the intervention were adequate. Completeness was positively associated with acceptability. Furthermore, positive effects (ie, dose-response effects) were found for completeness and time of website use on the mothers’ IDM, decisional conflict, and almost all of the social-psychological determinants of HPV vaccination acceptability. In addition, the extent to which mothers completed the intervention had a positive impact on their daughters’ vaccination uptake. This indicates that the web-based, tailored intervention fits well with the mothers’ needs, and that completeness of use is essential for improving HPV vaccination uptake, acceptability, and IDM. Program use should therefore be promoted. Trial Registration: Netherlands Trial Register NTR4795; https://www.trialregister.nl/trial/4795
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