Several cross-sectional studies have shown hair cortisol concentration to be associated with adiposity, but the relationship between hair cortisol concentration and longitudinal changes in measures of adiposity are largely unknown. We included 786 adults from the NoHoW trial, who had achieved a successful weight loss of ≥5% and had a body mass index of ≥25 kg/m2 prior to losing weight. Hair cortisol concentration (pg/mg hair) was measured at baseline and after 12 months. Body weight and body fat percentage were measured at baseline, 6-month, 12-month and 18-month visits. Participants weighed themselves at home ≥2 weekly using a Wi-Fi scale for the 18-month study duration, from which body weight variability was estimated using linear and non-linear approaches. Regression models were conducted to examine log hair cortisol concentration and change in log hair cortisol concentration as predictors of changes in body weight, change in body fat percentage and body weight variability. After adjustment for lifestyle and demographic factors, no associations between baseline log hair cortisol concentration and outcome measures were observed. Similar results were seen when analysing the association between 12-month concurrent development in log hair cortisol concentration and outcomes. However, an initial 12-month increase in log hair cortisol concentration was associated with a higher subsequent body weight variability between month 12 and 18, based on deviations from a nonlinear trend (b: 0.02% per unit increase in log hair cortisol concentration [95% CI: 0.00, 0.04]; P=0.016). Our data suggest that an association between hair cortisol concentration and subsequent change in body weight or body fat percentage is absent or marginal, but that an increase in hair cortisol concentration during a 12-month weight loss maintenance effort may predict a slightly higher subsequent 6-months body weight variability. Clinical Trial Registration: ISRCTN registry, identifier ISRCTN88405328.