A placebo-controlled, depletion-repletion protocol was utilized to examine the effect of vitamin C status on substrate utilization during a 90 min walk at 50% maximal oxygen consumption (VO2max). Nine vitamin C depleted subjects (plasma vitamin C < 28 mumol/L) agreed to participate in the 5-week study (aged, 27.6 +/- 2.5 years, mean +/- SE; 5 females, 4 males). Subjects were apparently healthy but unaware of their vitamin C status. Prior to the experimental period, VO2max was measured using open-circuit spirometry during a graded walking protocol. Subjects ingested a placebo capsule daily during weeks 1-3 and a 500 mg vitamin C capsule daily during weeks 4-5 of the experimental study. Mean plasma vitamin C rose nearly 3-fold and mean plasma carnitine fell by nearly 20% at repletion (week 5) versus depletion (week 3). At the end of weeks 3 and 5, subjects completed a 90 minute treadmill walk at an exercise intensity of 50% VO2max. The relative contribution of fat utilized for energy during walking did not differ in the vitamin C depleted versus repleted states. However, work performed by subjects and gross efficiency during exercise increased significantly at repletion versus depletion (10% and 15%, respectively). These data indicate that vitamin C depletion is associated with reduced work efficiency during submaximal exercise.