BACKGROUND: Aspergillus niger prolyl endoprotease (AN-PEP) efficiently degrades gluten molecules into non-immunogenic peptides in vitro. AIM: To assess the efficacy of AN-PEP on gluten degradation in a low and high calorie meal in healthy subjects. METHODS: In this randomised, double-blind, placebo-controlled, cross-over study 12 healthy volunteers attended to four test days. A liquid low or high calorie meal (4 g gluten) with AN-PEP or placebo was administered into the stomach. Via a triple-lumen catheter gastric and duodenal aspirates were sampled, and polyethylene glycol (PEG)-3350 was continuously infused. Acetaminophen in the meals tracked gastric emptying time. Gastric and duodenal samples were used to calculate 240-min area under the curve (AUC0-240 min ) of ?-gliadin concentrations. Absolute ?-gliadin AUC0-240 min was calculated using duodenal PEG-3350 concentrations. RESULTS: AN-PEP lowered alpha-gliadin concentration AUC0-240 min, compared to placebo, from low and high calorie meals in stomach (low: 35 vs. 389 mug x min/mL; high: 53 vs. 386 mug x min/mL; P < 0.001) and duodenum (low: 7 vs. 168 mug x min/mL; high: 4 vs. 32 mug x min/mL; P < 0.001) and absolute alpha-gliadin AUC0-240 min in the duodenum from low (2813 vs. 31 952 mug x min; P < 0.001) and high (2553 vs. 13 095 mug x min; P = 0.013) calorie meals. In the placebo group, the high compared to low calorie meal slowed gastric emptying and lowered the duodenal alpha-gliadin concentration AUC0-240 min (32 vs. 168 mug x min/mL; P = 0.001). CONCLUSIONS: AN-PEP significantly enhanced gluten digestion in the stomach of healthy volunteers. Increasing caloric density prolonged gastric residence time of the meal. Since AN-PEP already degraded most gluten from low calorie meals, no incremental effect was observed by increasing meal caloric density. ClinicalTrials.gov, Number: NCT01335503; www.trialregister.nl, Number: NTR2780.