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Introduction: A major component of the digesta reaching the colon from the distal ileum is carbohydrate. This carbohydrate is subject to microbial fermentation and can radically change bacterial populations in the colon and the metabolites they produce, particularly short-chain fatty acids (SCFA). However, very little is currently known about the forms and levels of carbohydrate in the ileum and the composition of the ileal microbiota in humans. Most of our current understanding of carbohydrate that is not absorbed by the small intestine comes from ileostomy models, which may not reflect the physiology of an intact gastrointestinal tract. Methods: We will investigate how ileal content changes depending on diet using a randomised crossover study in healthy humans. Participants will be inpatients at the research facility for three separate 4-day visits. During each visit, participants will consume one of three diets, which differ in carbohydrate quality: 1) low-fibre refined diet; 2) high-fibre diet with intact cellular structures; 3) high-fibre diet where the cellular structures have been disrupted (e.g. milling, blending). On day 1, a nasoenteric tube will be placed into the distal ileum and its position confirmed under fluoroscopy. Ileal samples will be collected via the nasoenteric tube and metabolically profiled, which will determine the amount and type of carbohydrate present, and the composition of the ileal microbiota will be measured. Blood samples will be collected to assess circulating hormones and metabolites. Stool samples will be collected to assess faecal microbiota composition. Subjective appetite measures will be collected using visual analogue scales. Breath hydrogen will be measured in real-time as a marker of intestinal fermentation. Finally, an in vitro continuous fermentation model will be inoculated with ileal fluid in order to understand the shift in microbial composition and SCFA produced in the colon following the different diets. Registration: ISRCTN11327221., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Byrne CS et al.)