A significant proportion of the population does not tolerate gluten. Aspergillus niger prolyl endopeptidase (AN-PEP) enzyme efficiently degrades gluten molecules into non-immunogenic peptides in a dynamic, multi-compartmental gastrointestinal simulation model but its efficacy in vivo remains to be established. Aim of our study was to assess the efficacy of ANPEP on gastrointestinal breakdown of gluten in healthy subjects, as well as the effect of meal caloric density on AN-PEP efficiency. Methods: In this double-blind, randomized, placebo-controlled cross-over study 12 healthy subjects attended to four test days in random order. On each occasion they received a low (143 kCal) or high caloric (405 kCal) meal, containing 4g gluten, with AN-PEP or placebo via a triple-lumen nasoduodenal catheter. Acetaminophen was added to measure gastric emptying rate. One lumen, positioned in the stomach, was used for administration of the test meal and collection of gastric fluid. A second lumen was used for the continuous injection of the inert dilution marker polyethylene glycol 3350 (PEG3350) to enable the calculation of meal dilution by endogenous secretions, with the injection port positioned just distal to the pylorus. A third lumen, located at the tube tip 10 cm distal to the second port, was used for collection of duodenal content. Fluid samples were taken regularly during 4 hours after meal infusion. The presence of the DQ2.5glia-α3 epitopes was quantified using the Gluten-Tec® ELISA assay, which provides an accurate estimate of the actual α-gliadin content of the samples. Degradation of intact gluten proteins was measured by Western-Blot analysis. Duodenal PEG3350 concentrations and gastric acetaminophen concentrations were determined by high-performance liquid chromatography. The effect of AN-PEP on gluten degradation was measured by the difference in 240-minute Area Under the Curve (AUC) of relative and absolute gluten exposure between AN-PEP and placebo. Differences between combinations of treatment and meal were assessed using linear mixed models. Results: AN-PEP significantly reduced gliadin concentration and absolute output in the stomach and duodenum (as 240-min AUCs) in both low and high caloric meals, compared to placebo. Furthermore, in the high caloric meal with placebo the duodenal gliadin concentration was lower, compared to the low caloric meal with placebo. No differences were observed between gliadin concentrations of a low or high caloric meal containing AN-PEP (Table). The gastric emptying time of the high caloric meal was longer compared to the low caloric meal, both in the presence of placebo (p=0.014) and AN-PEP (p=0.100). Conclusion: AN-PEP addition to a gluten containing meal significantly enhances gluten digestion in healthy volunteers. Meal caloric density does not affect the efficacy of AN-PEP on gluten degradation. Gliadin concentration and absolute output in stomach and duodenum