Peak brachial artery dilation post-nitroglycerin (NTG) administration occurs between 3 and 5-min in adults. The purpose of this study was to identify the time to peak dilation response to sublingual NTG (0.3 mg) in youth. Endothelium-independent dilation (EID) was measured in 198 healthy (113 males, 85 females) youth (6-18 yrs) via ultrasound imaging of the brachial artery following NTG administration up to 5-min. Time to peak EID was 4-min, 28-sec, following NTG administration. There was a significant (p<0.001) difference post-NTG at the 3 vs. 4-min, 4 vs. 5-min, and 3 vs. 5-min time points. Peak EID (males: 24.8 + 0.5 vs. females: 25.3 + 0.6 %, p=0.6) was not significantly different between genders after adjusting for baseline brachial diameter. Endothelium-independent dilation was measured up to 8-min in a small sub-group of 20 youth (12 males, 8 females), resulting in a time to peak dilation of 4-min, 24-sec, and an average change in peak dilation of 23.8 + 1.2 %. No significant difference existed beyond the 4-min time point within the small sub-group. In conclusion, peak response to NTG administration occurred between 4 and 5-min. The results demonstrate the importance of measuring EID up to 5-min post-nitroglycerin administration in youth.