Flow-mediated dilation (FMD) has been established as a reliable noninvasive measurement of endothelial function. The reproducibility of FMD under resting conditions has previously been reported; however, the reproducibility of FMD in response to exercise remains to be investigated. On two separate days, we determined if flow-mediated dilation is reproducible in response to acute exercise in nine overweight men. Following pre-exercise FMD measurements, subjects were asked to walk on a treadmill for 45 min at 50% of their VO(2)peak. Subsequently, FMD was measured immediately, and every hour for 3 h thereafter. Reproducibility of FMD following exercise was assessed utilizing: (1) a two-way analysis of variance (ANOVA), (2) Intraclass correlation coefficients (ICC), (3) Pearson correlations (r), and (4) coefficient of variation (CV %) and coefficient of variation prime (CV') for FMD at each time-period. Four acceptable reproducibility assessments were required to confirm FMD reproducibility in response to acute exercise. No differences (F(1,8) = .01; p = 0.942) in FMD were observed between trials collapsing for time. All the ICC(FMD) fell within the reproducible criterion set and are as follows: pre-exercise 0.602, immediately post 0.840, 1 h post 0.632, 2 h post 0.724 and 3 h post 0.631. The correlation and the average CV% for FMD between trials was 0.579% and 25.2%, respectively. The FMD response to an acute bout of moderate treadmill exercise appears to be as reproducible as FMD measured during resting conditions. The findings of the present study support the use of FMD as an outcome variable in response to acute exercise.