Self-applied and professional fluorides are key elements to limit caries-related side-effects during orthodontic treatment with fixed appliances. To evaluate the effectiveness of a new fluoride varnish formula containing 1.5% ammonium fluoride in preventing white spot lesions (WSLs) in adolescents undergoing multi-bracket orthodontic treatment. The study employed a randomized controlled triple-blinded design with two parallel arms. One hundred eighty-two healthy adolescents (12-18 years) referred to three orthodontic specialist clinics were eligible and consecutively enrolled. Informed consent was obtained from 166 patients and they were randomly allocated to a test or a placebo group (with aid of a computer program, generating sequence numbers in blocks of 15). In the test group, fluoride varnish was applied in a thin layer around the bracket base every sixth week during the orthodontic treatment, while patients in the placebo group received a varnish without fluoride. The intervention started at onset of the fixed appliances and continued until debonding. The endpoint was prevalence and severity of WSLs on the labial surfaces of the maxillary incisors, canines, and premolars as scored from high-resolution pre- and post-treatment digital photos with aid of a four-level score. One hundred forty-eight patients completed the trial, 75 in the test group and 73 in the placebo group (dropout rate 10.8%). The total prevalence of WSL's on subject level after debonding was 41.8% in the test group and 43.8% in the placebo group. The number of patients exhibiting more severe lesions (score 3 + 4) was higher in the placebo group (P < 0.05); the absolute risk reduction was 14% and the number needed to treat was 7.1. The multicentre design with somewhat diverging routines at the different clinics may have increased risk for performance bias. No health-economic evaluation was carried out. Regular applications of an ammonium fluoride varnish reduced the prevalence of advanced WSL during treatment with fixed orthodontic appliances. ClinicalTrials.gov (NCT03725020). The protocol was not published before trial commencement.