The gluten proteins of wheat grain are responsible for visco-elastic properties of flour, but they also trigger the immune-response of celiac disease. In this work, two low-gliadin RNA interference (RNAi) wheat lines that differ for the promoter driving the silencing (D-hordein and γ-gliadin promoters for D783 and D793 lines, respectively), were characterized at transcriptomic, and protein fraction levels in the grain. Silencing of gliadins provides a readjustment in the grain protein fractions that also affects to the non-gluten proteins (NGP), which were increased in both RNAi lines. Determination of wheat gluten by means of the R5 monoclonal antibody also showed a strong reduction in the content of gluten in both RNAi lines. Moreover, fructans, an oligosaccharide linked with the development of non-celiac wheat sensitivity (NCWS) were also significantly decreased in RNAi lines. The down-regulation of gliadins fractions also impacts to other metabolic processes, particularly on carbohydrate metabolism, enzyme regulator activity and response to stress. Genes and transcription factors regulated by ABA were up-regulated, which could suggest the implication of this phytohormone on the stress response observed in the RNAi lines. This research was funded by the Spanish Ministry of Science and Innovation (Project PID2019-110847RB-I00) and the European Regional Development Fund (FEDER).