Background: Circadian rhythms play an important role in the regulation of eating and fasting, and mistimed dietary intakes may be detrimental to metabolic health. Extended overnight fasting has been proposed as a strategy to better align the eating-fasting cycle with the internal circadian clock, and both observational and experimental studies have linked longer overnight fasting with lower body weight. However, it remains unclear if the timing of overnight fasting modifies the relationship between fasting duration and weight outcomes. Methods: The current study included 495 men and 499 women age 50–74 years. Dietary intake over 12 months was assessed by 24-h dietary recalls every two months, and body-mass index was measured at the beginning, middle and end of the study. Logistic regression was used to estimate the relationship between overnight fasting duration and the likelihood of being overweight or obesity adjusted for multiple confounders, and assessed whether the relationship was modified by the timing of overnight fasting, measured as the midpoint of the fasting period. Results: Among participants with early overnight fasting (midpoint < 02:19 am), a longer fasting duration was associated with lower odds of overweight and obesity; while among those with late fasting (≥02:19 am), longer fasting was associated with higher odds of overweight and obesity. Specifically, when compared to the shortest quintile of overnight fasting duration, the longest quintile was associated with a 53% reduction in the odds of overweight and obesity in the early fasting group (OR = 0.47, 95% CI = 0.23, 0.97), but a 2.36-fold increase in the late fasting group (OR = 3.36, 95% CI = 1.48, 7.62). Additionally adjusting for dietary intakes during morning and late evening periods did not affect the observed associations. Conclusions: Longer overnight fasting was associated with a reduced likelihood of being overweight or obese, but only among those with an early timing of fasting.