Purpose Nuclear Overhauser effect (NOE) is based on dipolar cross‐relaxation mechanism that enables the indirect detection of aliphatic protons via the water proton signal. This work focuses on determining the reproducibility of NOE magnetization transfer ratio (NOEMTR) and isolated or relayed NOE (rNOE) contributions to the NOE MRI of the healthy human brain at 7 Tesla (T). Methods We optimized the B1+$$ {\mathrm{B}}_1^{+} $$ amplitude and length of the saturation pulse by acquiring NOE images with different B1+$$ {\mathrm{B}}_1^{+} $$ values with multiple saturation lengths. Repeated NOE MRI measurements were made on five healthy volunteers by using optimized saturation pulse parameters including correction of B0 and B1+$$ {\mathrm{B}}_1^{+} $$ inhomogeneities. To isolate the individual contributions from z‐spectra, we have fit the NOE z‐spectra using multiple Lorentzians and calculated the total contribution from each pool contributing to the overall NOEMTR contrast. Results We found that a saturation amplitude of 0.72 μT and a length of 3 s provided the highest contrast. We found that the mean NOEMTR value in gray matter (GM) was 26%, and in white matter (WM) was 33.3% across the 3D slab of the brain. The mean rNOE contributions from GM and WM values were 8.9% and 9.6%, which were ∼10% of the corresponding total NOEMTR signal. The intersubject coefficient of variations (CoVs) of NOEMTR from GM and WM were 4.5% and 6.5%, respectively, whereas the CoVs of rNOE were 4.8% and 5.6%, respectively. The intrasubject CoVs of the NOEMTR range was 2.1%–4.2%, and rNOE range was 2.9%–10.5%. Conclusion This work has demonstrated an excellent reproducibility of both inter‐ and intrasubject NOEMTR and rNOE metrics in healthy human brains at 7 T.