The most widely employed methodologies for type III (retrograded) resistant starch formation is a cyclic heating-cooling treatment or its combination with a previous enzymatic debranching step. In this work, two retrogradation temperatures, 0 and 4 °C, were evaluated for obtaining type III resistant starch from non-debranched and pullulanase-debranched wheat starch. The starches were characterized by their thermal and pasting behavior. Also, microstructural and crystalline aspects were related to the production process and the nutritional properties of the products. The most influencing factor for resistant starch formation was the application of the pullulanase-debranching step previous to the cyclic treatment. This led to a higher starch retrogradation and a more compact microstructure, which decreased the thickening capacity and the digestibility of the starches. Then, even though all the obtained starches exhibited lower digestibility, the pullulanase-debranched ones presented the lowest estimated glycemic index (ca. 34%), showing the highest yield of resistant starch formation. Finally, this work presents a concise protocol for obtaining type III resistant starch enriched powder with low digestibility that could be used in the formulation of different healthy foods such as dairy products, cereal bars, and even drinks.Centro de Investigación y Desarrollo en Criotecnología de Alimentos