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The goal of this work was to systematically evaluate the reproducibility of amide proton transfer chemical exchange saturation transfer (APT-CEST) at 3 T and its signal dependence on pH, protein concentration, and acquisition parameters. An in vitro system based on bovine serum albumin (BSA) was used, and its limitations were tested by comparing it to in vivo measurements. The contribution of small endogenous metabolites on the APT-CEST signal at 3 T was also investigated. In addition, the reliability of different z-spectrum interpolations as well as the use of only a few frequency offset data points instead of a whole z-spectrum were tested.We created both a BSA phantom at different concentrations and pH values and a metabolite phantom with different small molecules. Chemical exchange saturation transfer data were acquired using a 2-dimensional fast spoiled gradient-echo sequence with pulsed CEST preparation at different saturation durations and power levels. Healthy volunteer measurements were taken for comparison. Z-spectra were interpolated using a 24th-order polynomial (Poly), an eighth-order Fourier series (Fourier), and a smoothing Spline (sSpline) algorithm. To evaluate reduced data sets, only 6 to 14 frequency offsets of the z-spectrum were used and interpolated via a cubic Spline. Region of interest (ROI) evaluations were used to investigate the reproducibility of amide magnetization transfer ratio asymmetry [MTRasym(3.5 ppm)] and to analyze the MTRasym and z-spectra.Interscan standard deviations of MTRasym(3.5 ppm) were always below 0.3%. MTRasym(3.5 ppm) increased when the BSA concentrations increased and decreased when the pH increased. The amine MTRasym signal of small molecules was very small compared with BSA and was only detectable using short saturation times and higher power levels. The MTRasym(3.5 ppm) between BSA concentration steps and between nearly all pH steps was significantly different for all 3 fitting methods. The Fourier and sSpline methods showed no statistically significant differences; however, the results for the Poly method were significantly higher at some concentrations and pH values. Using only few frequency offsets resulted in less significant differences compared with fitting the complete z-spectrum. In general, MTRasym(3.5 ppm) of gray matter, white matter, and ventricle ROIs from volunteer scans increased with an increase in saturation power and partially decreased with an increase in saturation duration. Intra-ROI covariances of MTRasym(3.5 ppm) revealed the highest variations for Poly, whereas using reduced spectral data resulted in an increased signal variation.Amide proton transfer–CEST imaging is a highly reproducible method in which absolute signal differences of approximately 0.5% are detectable in principle. For in vivo applications, Fourier or sSpline interpolations of z-spectra are preferable. Using reduced data sets delivers similar results but with increased variation and therefore decreased (pH/concentration) differentiation capability. Differentiation capability increases with increases in the saturation duration and power level. The results from the in vitro BSA system cannot be directly transferred to the in vivo situation due to different chemical environments resulting in, for example, higher asymmetric macromolecular cMT effects in vivo. Amine signals from small molecules are unlikely to contribute to APT-CEST at 3 T (except for creatine); however, signals can be enhanced by using short saturation times and higher power levels.