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Background Several studies have suggested that reduced sleep duration and quality are associated with an increased risk of obesity and related metabolic disorders, but the role of sleep in long-term weight loss maintenance (WLM) has not been thoroughly explored using prospective data. Methods and findings The present study is an ancillary study based on data collected on participants from the Navigating to a Healthy Weight (NoHoW) trial, for which the aim was to test the efficacy of an evidence-based digital toolkit, targeting self-regulation, motivation, and emotion regulation, on WLM among 1,627 British, Danish, and Portuguese adults. Before enrolment, participants had achieved a weight loss of [greater than or equal to]5% and had a BMI of [greater than or equal to]25 kg/m.sup.2 prior to losing weight. Participants were enrolled between March 2017 and March 2018 and followed during the subsequent 12-month period for change in weight (primary trial outcome), body composition, metabolic markers, diet, physical activity, sleep, and psychological mediators/moderators of WLM (secondary trial outcomes). For the present study, a total of 967 NoHoW participants were included, of which 69.6% were women, the mean age was 45.8 years (SD 11.5), the mean baseline BMI was 29.5 kg/m.sup.2 (SD 5.1), and the mean weight loss prior to baseline assessments was 11.4 kg (SD 6.4). Objectively measured sleep was collected using the Fitbit Charge 2 (FC2), from which sleep duration, sleep duration variability, sleep onset, and sleep onset variability were assessed across 14 days close to baseline examinations. The primary outcomes were 12-month changes in body weight (BW) and body fat percentage (BF%). The secondary outcomes were 12-month changes in obesity-related metabolic markers (blood pressure, low- and high-density lipoproteins [LDL and HDL], triglycerides [TGs], and glycated haemoglobin [HbA1c]). Analysis of covariance and multivariate linear regressions were conducted with sleep-related variables as explanatory and subsequent changes in BW, BF%, and metabolic markers as response variables. We found no evidence that sleep duration, sleep duration variability, or sleep onset were associated with 12-month weight regain or change in BF%. A higher between-day variability in sleep onset, assessed using the standard deviation across all nights recorded, was associated with weight regain (0.55 kg per hour [95% CI 0.10 to 0.99]; P = 0.016) and an increase in BF% (0.41% per hour [95% CI 0.04 to 0.78]; P = 0.031). Analyses of the secondary outcomes showed that a higher between-day variability in sleep duration was associated with an increase in HbA1c (0.02% per hour [95% CI 0.00 to 0.05]; P = 0.045). Participants with a sleep onset between 19:00 and 22:00 had the greatest reduction in diastolic blood pressure (DBP) (P = 0.02) but also the most pronounced increase in TGs (P = 0.03). The main limitation of this study is the observational design. Hence, the observed associations do not necessarily reflect causal effects. Conclusion Our results suggest that maintaining a consistent sleep onset is associated with improved WLM and body composition. Sleep onset and variability in sleep duration may be associated with subsequent change in different obesity-related metabolic markers, but due to multiple-testing, the secondary exploratory outcomes should be interpreted cautiously. Trial registration The trial was registered with the ISRCTN registry (ISRCTN88405328)., Author(s): Sofus C. Larsen 1,*, Graham Horgan 2, Marie-Louise K. Mikkelsen 1, Antonio L. Palmeira 3, Sarah Scott 4, Cristiana Duarte 4, Inês Santos 3,5, Jorge Encantado 3, Ruairi O'Driscoll [...]