Purpose: Metabolic MRI is a noninvasive technique that can give new insights into understanding cancer metabolism and finding biomarkers to evaluate or monitor treatment plans. Using this technique, a previous study has shown an increase in pH during neoadjuvant chemotherapy (NAC) treatment, while recent observation in a different study showed a reduced amide proton transfer (APT) signal during NAC treatment (negative relation). These findings are counterintuitive, given the known intrinsic positive relation of APT signal to pH. Methods: In this study we combined APT MRI and 31P-MRSI measurements to unravel the relation between the APT signal and pH in breast cancer. Twenty-two breast cancer patients were scanned with a 7 T MRI before and after the first cycle of NAC treatment. pH was determined by the chemical shift of inorganic phosphate (Pi). Results: While APT signals have a positive relation to pH and amide content, we observed a direct negative linear correlation between APT signals and pH in breast tumors in vivo. Conclusions: As differentiation of cancer stages was confirmed by observation of a linear correlation between cell proliferation marker PE/Pi (phosphoethanolamine over inorganic phosphate) and pH in the tumor, our data demonstrates that the concentration of mobile proteins likely supersedes the contribution of the exchange rate to the APT signal.