Abstract Functional magnetic resonance imaging (fMRI) is a widely used tool to investigate the functional brain responses in living humans. Valid comparisons of fMRI results depend on consistency of the blood-oxygen-level-dependent (BOLD) hemodynamic response function (HRF). Although common statistical approaches assume a single HRF across the entire brain, the HRF differs across individuals, regions of the brain, and cortical depth. Here, we measure HRF properties in primary visual cortex (V1) using 7 T fMRI with ultra-high spatiotemporal resolution line-scanning (250 μm in laminar direction, sampled every 105 ms). Line-scanning allowed us to investigate age-related HRF changes as a function of cortical depth. Eleven young and eleven middle-aged healthy participants participated in the experiments. We estimated the HRFs using a smooth basis function deconvolution approach. We also compared the results with conventional resolutions. From these HRFs, we extracted properties related to response magnitude and temporal dynamics. The cortical depth dependent HRFs were similar to the HRFs extracted using conventional resolutions validating the cortical depth dependent approach. We found that the properties of the HRF in the two age groups are similar across cortical depth. In other words, the variance between participants is larger than the variance between age groups. This suggests that middle-aged individuals can participate in cortical depth dependent studies free of bias in HRF properties.