Mastication initiates digestion, disintegrating food structure and mixing it with saliva. This study aimed to provide understanding about the first step of bread digestion by exploring release of compounds from bread matrix during mastication. Furthermore, the aim was to identify compound groups that differentiate rye and wheat breads. Fifteen participants masticated whole-meal rye bread, endosperm rye bread, endosperm rye bread with added gluten and wheat bread. The masticated samples were studied with non-targeted LC-MS metabolic profiling. A great number of compounds were released from bread matrices in mastication, and the identified compounds differed largely between bread types. Specifically, rye bread samples were characterized by a greater release of peptides and amino acids, whereas sugars and nucleosides were characteristic for wheat bread. These compounds could potentially act as signal molecules in the alimentary tract and may explain, at least partly, the postprandial physiological effects of the breads identified in earlier studies.