Chemical Exchange Saturation Transfer (CEST) imaging can generate contrast that is sensitive to amide protons associated with proteins and peptides (termed amide proton transfer, APT). In breast cancer, APT contrast may report on underlying changes in microstructural tissue composition. However, to date, there have been no developments or applications of APT CEST to breast cancer. As a result, the aims of this study were to i) experimentally explore optimal scan parameters for breast CEST near the amide resonance at 3 T, ii) establish the reliability of APT imaging of healthy fibroglandular tissue, and iii) demonstrate preliminary results on APT changes in locally advanced breast cancer observed during the course of neoadjuvant chemotherapy. CEST measurements were experimentally optimized on cross-linked bovine serum albumin phantoms and the reliability of APT imaging was assessed in ten women with no history of breast disease. The mean difference between test-retest APT values was not significantly different from zero, and the individual difference values were not dependent upon the average APT value. The 95% confidence interval limits were ± 0.70% (α = 0.05) and the repeatability was 1.91. APT measurements were also performed in three women before and after one cycle of chemotherapy. Following therapy, APT increased in the one patient with progressive disease and decreased in the two patients with a partial or complete response. Together, these results suggest that APT imaging may report on treatment response in these patients.