Previous research suggests that vitamin C depletion modifies respiratory exchange ratio (RER) and, in particular, fat oxidation in healthy, normal-weight individuals. Impaired fat oxidation has been implicated in the development of obesity and failed weight loss attempts. The purpose of this study was to assess the effect of vitamin C supplementation on fat oxidation and fatigue (Ratings of Perceived Exertion, RPE) during a 50-minute walk at 40 percent of estimated maximal oxygen consumption (VO2max) using a randomized, double-blinded, placebo-controlled, 4-week intervention trial in vitamin C-depleted overweight men. Fifteen relatively sedentary, overweight (Body Mass Index, BMI, greater than 25) men (mean age 30.7 years) unaware of their vitamin C status volunteered to participate. Subjects were instructed to follow a vitamin C poor diet for a three-week depletion period and were randomly assigned to either placebo (PL, 8 subjects) or vitamin C (VC: 1 gram/day, 7 subjects) for the 4-week intervention. Independent t-tests and repeated measures analysis of variance were used in statistical analyses; significance was placed for p-values less than 0.05. After the 4-week intervention, plasma vitamin C concentrations increased from baseline in VC but did not change in PL (p-value was 0.019 for time x group effect). There were no significant differences in measures of body weight, fat mass, BMI, physical activity, or VO2max. Exercise RER decreased in both groups and nearly reached significance in PL; however, after adjusting for the effects of change in body weight and physical activity this trend was no longer observed. No significant differences between groups were noted during the 50-minute walk in any of the exercise indices. In summary, vitamin C did not appear to stimulate fat oxidation or lower RPE in free-living, vitamin C-depleted, overweight men during submaximal exercise. Modest acute changes in body weight and physical activity may have a greater role in eliciting beneficial alterations of substrate utilization.