Gluten and amylase/trypsin-inhibitors (ATIs) are known triggers for wheat-related disorders. Ancient wheat species like einkorn, emmer, Khorasan wheat, Polish wheat and spelt are suggested to have fewer of these immunoreactive proteins compared to modern common wheat, because breeding for higher yields and increased resistance to plant diseases might have resulted in changes in the protein composition. Currently, there are only very few studies on the protein composition of ancient wheat species. This comparative study investigated the bread volume, protein content as well as gluten and ATI content and composition, along with the inhibitory activity in ten ancient wheat varieties cultivated under organic conditions. The varieties had different ploidy levels and were harvested in three consecutive years. The various analyses consistently concluded that differences in protein composition were associated with the different ploidy levels. This means that the results were similar for the diploid species (einkorn), the tetraploid species (emmer, durum wheat, Khorasan wheat and Polish wheat) and for the hexaploid species (spelt and common wheat), respectively. These results suggest that breeding has likely not increased the immunoreactive potential of wheat and ancient wheats are unlikely to provide improved tolerability for individuals with wheat-related disorders.