It has been deemed important to normalize flow-mediated dilation (FMD), a marker of endothelial function, for between-subject differences in the eliciting shear-rate (SR) stimulus. Conventionally, FMD is divided by the area-under-the-curve of the shear-rate stimulus. In the context of a cross-sectional comparison across different age cohorts, we examined whether this ratio approach adhered to established statistical assumptions necessary for reliable normalization. In order to quantify brachial artery FMD and area-under-the-curve of SR, forearm cuff inflation to supra-systolic pressure was administered for 5 min to 16 boys aged 10.9 years (SD 0.3), 48 young men aged 25.3 years (SD 4.2) and 15 older men aged 57.5 years (SD 4.3). Mean differences between age-groups were statistically significant (P<0.001) for non-normalized FMD (children: 10.4% (SD 5.4), young adults: 7.5% (SD 2.9), older adults: 5.6% (SD 2.0), but not for ratio-normalized FMD (P=0.10). Moreover, all assumptions necessary for reliable use of ratio-normalization were violated, including regression slopes between SR and FMD that had y-intercepts greater than zero (P<0.05), non-linear and unstable relations between the normalized ratios and SR, skewed data distributions and heteroscedastic variance. Logarithmic transformation of SR and FMD prior to ratio calculation improved adherence to these assumptions and resulted in age-differences similar to the non-normalized data (P=0.03). In conclusion, although ratio-normalization of FMD altered findings about age-differences in endothelial function, this could be explained by violation of statistical assumptions. We recommend that exploration of these assumptions should be routine in future research. If the relationship between shear rate and FMD is generally found to be weak or non-linear or variable between samples, then ratio normalization should not be applied.