BACKGROUND: Topical fluorides are commonly recommended to prevent the development of white spot lesion (WSL) during treatment with fixed orthodontic appliances (FOAs), but the certainty of evidence is low, and long-term effects of fluoride preventive methods to reduce lesions seem to be rare. OBJECTIVE: To evaluate the long-term effectiveness of professional applications of a fluoride varnish containing 1.5% ammonium fluoride in preventing WSL development in adolescents undergoing multi-bracket orthodontic treatment. SUBJECTS AND METHODS: We performed a randomized controlled trial in which 166 healthy adolescents (12-18 years) from three different clinics were enrolled and randomly allocated to a test or a placebo group. The randomization was performed by a computer program, generating sequence numbers in blocks of 15. The fluoride varnish or the non-fluoride placebo varnish was applied in a thin layer around the bracket base every sixth week during the course of the orthodontic treatment (mean duration 1.7 years). We scored the prevalence of WSL on the labial surfaces of the maxillary incisors, canines and premolars immediately after debonding (baseline) and approximately 1 year after debonding, from digital photos with aid of a four-step score. The examiners were not involved in the treatment of the patients and blinded for the group assignment. RESULTS: One hundred and forty-eight patients were available at debonding and 142 of them could be re-examined after 1 year (71 in the test and 71 in the placebo group). The 1-year attrition rate was 4.0%. On patient level, the prevalence of post-orthodontic WSLs (score ≥ 2) dropped by over 50% during the follow-up with no significant difference between the groups. On surface level, there were significantly fewer remaining WSLs in the test group compared with the placebo group (4.5% versus 10.4%; relative risk 0.44, 95% confidence interval 0.28-0.68). LIMITATIONS: The compliance with fluoride toothpaste was not checked, and the patients' general dentists may have instigated additional risk-based preventive measures. No cost-benefit analysis was carried out. CONCLUSIONS: This follow-up study displayed a small beneficial long-term effect of fluoride varnish in reducing WSL development during treatment with FOA. REGISTRATION: NCT03725020. PROTOCOL: The protocol was not published before trial commencement.