Background: In wheat-dependent exercise-induced anaphylaxis (WDEIA), cofactors such as exercise, acetylsalicylic acid (ASA), alcohol or unfavorable climatic conditions are required to elicit a reaction to wheat products. The mechanism of action of these cofactors is unknown, but an increase of gliadin absorption has been speculated. Our objectives were to study gliadin absorption with and without cofactors and to correlate plasma gliadin levels with factors influencing protein absorption in healthy volunteers. Methods: Twelve healthy probands (six males, six females; aged 20-56 years) ingested 32 g of gluten without any cofactor or in combination with cofactors aerobic and anaerobic exercise, ASA, alcohol and pantoprazole. Gliadin serum levels were measured up to 120 min afterwards and the intestinal barrier function protein zonulin in stool was collected before and after the procedure; both were measured by ELISA. Stool microbiota profile was obtained by 16S gene sequencing. Results: Within 15 min after gluten intake, gliadin concentrations in blood serum increased from baseline in all subjects reaching highly variable peak levels after 15-90 min. Addition of cofactors did not lead to substantially higher gliadin levels, although variability of levels was higher with differences between individuals (p < 0.001) and increased levels at later time points. Zonulin levels in stool were associated neither with addition of cofactors nor with peak gliadin concentrations. There were no differences in gut microbiota between the different interventions, although the composition of microbiota (p < 0.001) and the redundancy discriminant analysis (p < 0.007) differed in probands with low versus high stool zonulin levels. Conclusion: The adsorption of gliadin in the gut in healthy volunteers is less dependent on cofactors than has been hypothesized. Patients with WDEIA may have a predisposition needed for the additional effect of cofactors, e.g., hyperresponsive or damaged intestinal epithelium. Alternatively, other mechanisms, such as cofactor-induced blood flow redistribution, increased activity of tissue transglutaminase, or increases in plasma osmolality and acidosis inducing basophil and mast cell histamine release may play the major role in WDEIA.