Purpose: No previous study has explored the importance of exercise-induced changes in vascular function to prolonged adaptations. Therefore, the purpose was to explore the within-subject relationship between the acute post-exercise change in brachial artery endothelial function (flow-mediated dilation, FMD) and the change in resting FMD after a 2-week exercise training in healthy volunteers. Methods: Twenty one healthy, young men (24 ± 5 years) underwent assessment of brachial artery FMD using high-resolution ultrasound before and after 30-min of moderate-intensity cycle exercise (80% maximal heart rate). Subsequently, subjects performed five 30-min cycle exercise bouts at 80% maximal heart rate across a 2-week period, followed by repeat assessment of resting brachial FMD post-training. Results: Correcting for changes in diameter and shear, FMD did not change after the initial exercise bout (P = 0.26). However, a significant correlation was found between post-exercise changes in FMD and adaptation in resting FMD after training (r = 0.634, P = 0.002), where an acute decrease in post-exercise FMD resulted in a decrease in baseline FMD after 2 weeks and vice versa. We also found a positive correlation between antegrade shear rate during exercise and change in FMD% after acute exercise and after exercise training (r = 0.529 and 0.475, both P < 0.05). Conclusion: Our findings suggest that acute post-exercise changes in vascular function are related to changes in resting FMD after a 2-week endurance exercise training period in healthy men, an effect that may be related to exercise-induced increases in antegrade shear rate. This provides further insight into the relevance of acute changes in shear and FMD for subsequent adaptation.