Food-dependent exercise-induced anaphylaxis is a unique form of food allergy. Recent studies indicate that gliadin is a major allergen in patients with wheat-dependent exercise-induced anaphylaxis (WDEIA). We evaluated the value of serum gliadin-specific IgE in the diagnosis of WDEIA, as well as the characteristics and digestibilities of the IgE-binding components of gliadin. We analyzed 32 adult subjects sensitized to wheat; 26 were asymptomatic sensitizers and 6 had WDEIA. Wheat flour and gliadin-specific IgE levels were measured, and corresponding receiver operator characteristics (ROC) curves were determined. Patients with WDEIA had higher levels of gliadin-specific IgE than patients without WDEIA (p = 0.003). The area under the ROC curve for gliadin-specific IgE was 0.872, which suggested this assay could be used as a supplementary test for the diagnosis of WDEIA. IgE immunoblot analyses of reactions to wheat and gliadin extracts were compared both with and without simulated gastric fluid (SGF) and intestinal fluid incubation (SIF). All six WDEIA patients had high IgE binding signals to 50 kDa of gliadin extract on IgE immunoblots. This binding was suppressed by SGF, although minimal inhibition was noted with SIF incubation.