Melatonin is recommended as a first-line treatment in isolated REM sleep behavior disorder (iRBD), although no large patient group has been reported. To assess effects, time-course and confounding factors in the treatment of patients with iRBD using melatonin, 209 consecutive patients were included in this single-center, observational cohort study. 171 patients had taken melatonin according to our chronobiotic protocol (2mg, ≥ 6 months, always-at-the-same-clock-time, 10-11pm, corrected for chronotype), 13 had applied melatonin for about 1-3 months, 25 underwent mixed treatments. In total, 1529 clinical evaluations were performed, including Clinical Global Impression (CGI) and a newly developed RBD-symptom severity scale (Ikelos-RS), analyzed using linear mixed models. Validation of Ikelos-RS showed excellent inter-rater reliability (ρ=0.9, p<0.001), test-retest reliability (ρ=0.9, p<0.001) and convergent validity (ρ=0.9, p<0.001). With melatonin RBD-symptom severity gradually improved over the first 4 weeks of treatment (Ikelos-RS: 6.1 vs. 2.5; CGI-Severity: 5.7 vs. 3.2) and remained stably improved (mean follow-up 4.2±3.1yrs; range: 0.6-21.7yrs). Initial response was slowed to up to 3 months with melatonin suppressing (betablockers) or REM sleep spoiling co-medication (antidepressants) and failed with inadequately timed melatonin intake. When melatonin was discontinued after 6 months, symptoms remained stably improved (mean follow-up after discontinuation 4.9±2.5yrs; range: 0.6-9.2). When administered only 1-3 months, RBD-symptoms gradually returned. Without any melatonin, RBD-symptoms persisted and did not wear off over time. Clock-timed, low dose, long-term melatonin treatment in patients with iRBD appears to be associated with improvement of symptoms. The outlasting improvement over years questions a pure symptomatic effect. Clock-time dependency challenges existing prescription guidelines for melatonin.