INTRODUCTION: The colon is subjected to a plethora of potentially damaging agents against which its first line of defense is the protective mucus bilayer, consisting of a removable loosely adherent layer and a firmly adherent layer difficult to remove [1]. We have recently shown that the firmly adherent mucus layer acts as a physical barrier to the microbiota. The mucus bilayer also reduces shear stress to the mucosa [2]. The colonic microbiota and mucosal health has been linked to different constituents in the diet [3]. The aim of this study was to investigate if a high-fat diet with or without prebiotic bilberry fibers influence on the thickness and secretion of the colonic mucus bilayers. METHODS: Male Sprague-Dawley rats (80g) were given a diet consisting of either low-fat diet (LFD), high-fat diet (HFD), LFD with bilberry extract (LFD+BB) or HFD with bilberry extract (HFD+BB), for 8 weeks. On the last day of experiment, rats (n=8/group) were anesthetized (Inactin, 120 mg/kg) and operated on for in vivo measurements of mucus thickness. The distal part of the colon was opened up along the anti-mesenteric border and draped over a truncated cone with the luminal side up. A mucosal chamber with warm saline was placed over the mucosa. To ease visualization through the microscope, saline-suspended charcoal was dropped on the mucus gel. To perform measurements, micropipettes, held by a micromanipulator at an angle of 35-45 degrees to the cell surface, was pushed into the mucus gel. The distance (l) to the epithelial surface was measured with a “digimatic indicator”. Mucus thickness (T) was then calculated using the formula: T =l x sin a. Mucus thickness was measured at different intervals, before and after removing the loosely adherent mucus by suction. Distal colon tissue was paraffin-embedded, sectioned and PAS-stained to count the number of goblet cells. RESULTS: Here we have demonstrated that: (1) 8 weeks of HFD supplementation with or without BB leads to increased thickness of the firmly adherent mucus layer (e.g. the mean firmly adherent mucus thickness rats fed a HFD or a HFD+BB (84.2±1.2 and 82.7±0.7 µm) was significantly higher than the respective LFD and LFD+BB controls (78.7± 0.7 and 78.6±0.9; Students t-test p< 0.0002 and p<0.001); (2) the loosely adherent mucus thickness is only increased by a diet with a combination of bilberry extract and high-fat (e.g. the mean loosely adhesive mucus thickness in the rats fed HFD+BB (40.7±1.5 µm) was significantly higher than in rats fed a HFD only (31.3±3.2 µm; Students t-test p<0.05). (3) a pilot study indicate an increase in the number of goblet cells in rats fed HFD+BB and a decrease in rats fed HFD only. SUMMARY: High-fat diet, with our without bilberries, increase the protective firmly adherent mucus layer, while only a combination of high-fat diet and bilberries increase the thickness of the loosely adherent mucus layer.