α-Dicarbonyl compounds (α-DCs), such as glyoxal, methylglyoxal and 2,3-butanedione, are highly reactive substances occurring in thermally treated and fermented foods, that may react with amino and sulphydryl groups of side chains of proteins to form Maillard reaction end products, inducing a negative impact on the digestibility and on nutritional value of protein. In recent years the role of food derived α-DCs in gastroduodenal tract is under investigation to understand whether excess consumption of such dietary compounds might be a risk for human health. In this study the interactions between a mixture of glyoxal, methylglyoxal and 2,3-butanedione and the digestive enzymes (pepsin and pancreatin) were studied. The results showed that during gastroduodenal digestion α-DCs react with digestive enzymes to produce carbonylated proteins. Moreover, undigested and digested α-DC cytotoxicity against human cells, as well as their ability to inhibit the function of human enzymes responsible for DNA repair were shown.