Introduction Response rates in health research are declining, and low response rates could result in biased outcomes when population characteristics of participants systematically differ from the non-respondents. Few studies have examined key factors of non-response beyond demographic characteristics, such as behavioral and psychological factors. The aim of the current study was to identify predictors of non-response and loss to follow-up in a longitudinal sexual health study. Materials and methods A longitudinal cohort study (iMPaCT) was conducted from November 2016 to July 2018 among heterosexual STI clinic visitors aged 18–24 years. At four different time points in one year, data was collected on sexual behavior, psychological determinants and chlamydia infections. The national STI surveillance database provided data on demographic, behavioral and sexual health-related characteristics for non-respondents. Predictors of nonresponse at baseline and of loss to follow-up were identified using multivariable logistic regression analyses. Results In total, 13,658 STI clinic visitors were eligible to participate, of which 1,063 (8%) participated. Male gender, low/medium education level, young age ( 20 years) and having a non-Dutch migration background were significant predictors of non-response at baseline. Furthermore, non-respondents at baseline were more likely to report STI-related symptoms, to have been notified by a partner, to have had condomless sex, and to have had 2 partners in the past six months, compared to participants. Psychological predictors of loss to followup differed between STI clinic regions, but low perceived importance of health at baseline was associated with loss to follow-up in all regions. The baseline chlamydia positivity rate was significantly higher in the non-respondents (17%) compared to the participants (14%), but was not a predictor of loss to follow-up. Discussion Targeted recruitment aimed at underrepresented groups in the population based on demographic, behavioral and psychological characteristics, might be necessary to decrease loss to follow-up, and to prevent non-response bias in health research.