BackgroundLonger overnight fasting (ONF) is a potential strategy for weight control. While promising, the evidence from large population-based studies is limited. ObjectiveTo examine the association of self-reported ONF duration with three- and six-year weight change in the American Cancer Society’s Cancer Prevention Study-3 (CPS-3) prospective cohort. MethodsU.S. adult CPS-3 participants completed a 24-hour validated meal and snack timing and frequency grid (weekday and weekend) in 2015, from which weighted ONF hours were calculated. Participants reported body weight in 2015, 2018 and 2021. Three- and six-year weight change (kg, and % body weight) were assessed. ResultsAmong 104,420 mostly female (78.5%) participants aged 52.7 +/- 9.5 (SD) years followed for six years, a one hour increase in ONF length was associated with a small but statistically significant reduction in weight gain over three- and six-year periods (multivariable-adjusted mean difference in % body weight=-0.02, 95% CI -0.05-0.00, p=0.03 and -0.04, 95 % CI, -0.07 to -0.01, p=0.007, respectively). The mean difference of 6-year % reduction in weight gain was slightly greater among individuals with overweight (-0.05, 95% CI, -0.10 to 0.00, p=0.05) and obesity (-0.06, 95% CI, -0.12 to 0.01, p=0.08) compared to those with healthy BMI (-0.03, 95% CI -0.07 to 0.01, p=0.13) or underweight (0.16, 95% CI, -0.04 to 0.36, p=0.13, pinteraction<0.0001). Stronger associations were observed among those ≤55 y than 56+ (pinteraction=0.01), and those with higher waist circumference (pinteraction<0.0001) but not by sex or earlier/later fasting period. ConclusionsLonger ONF was associated with slightly lower body weight in adult men and women over six years that was stronger among those with overweight or obesity, higher waist circumference, and those ≤age 55. The magnitude of weight change, though in the hypothesized direction, suggests that prolonged ONF may have modest impact on weight control over time.