A diet with a high glycemic index (GI) is associated with an elevated risk for obesity or type 2 diabetes. We investigated the GI of a newly-developed fiber enriched cookie and characterized the microstructure of ingredients used. In a study with 26 non-diabetic healthy volunteers it was shown that the fiber enriched cookie has a GI of 58.9 in relation to white bread as reference. Using a conversion factor of 1.4, the GI of the fiber enriched cookie in relation to a glucose-solution is 42.0 and can be classified as a low-GI food. Postprandial insulin concentration was significantly lower after consumption of fiber enriched cookies compared to white bread. Glucose release after in vitro digestion was significantly lower from fiber enriched cookies compared to other cookies tested. In addition to its high percentage of fiber, the cookies’ low GI can be attributed to the limited gelatinization potential of the starch granules found in the ingredients used. Using confocal laser scanning microscopy it is shown that starch granule surface area of whole grain barley flour, spelt flour and oat flakes bears cluster-shaped protein–NSPS complexes that preferentially absorb water in conditions of water shortage and thereby prevent starch gelatinization.