Background: The present study aims to detect, quantify and analyze circulating nutritional antigen-specific T-cells in patients with celiac disease (CeD) as well as inflammatory bowel disease (IBD), thus comparing the specific T-cell response following barrier disruption and antigen translocation. Methods: The antigen-reactive T-cell enrichment (ARTE) technique was applied allowing for a phenotypical and functional flow cytometric analysis of rare nutritional antigen-specific T-cells, including the CeD-causing gliadin (gluten), in the peripheral blood. Results: Our study indicates that by applying the ARTE technique, differences of gluten-specific T-cells as well as the differential cytokine expression between the patient groups can be detected, even without the burdening gluten re-exposure of the patients. CeD patients, independent from the presence or absence of gluten exposure in their current diet, featured an increase of the frequency of gliadin-specific T-cells, which were characterized by a pro-inflammatory phenotype. However, only for active CeD and a consecutive small intestinal barrier breach, an increase of distinct nutritional T-cells could be detected. Accordingly, frequency as well as pro-inflammatory phenotype of nutritional antigen-specific T cells were highest in Crohn’s disease patients with small intestinal inflammation whereas no significant increase was observed in ulcerative colitis. Conclusion: In summary, the ARTE method allows not only for detection but also for functional analysis of these rare cells even in healthy subjects. Applying this method, we were able to demonstrate that for non-CeD-related nutritional antigens, small intestinal barrier breach is mandatory for a peripheral antigen-specific T-cell.