Nutrition science requires more science-based evidences for the development of effective functional diets. To reduce animals for experimental purposes innovative reliable and informative models, simulating the complex intestinal physiology, are needed. The aim of this study was to develop a swine duodenum segment perfusion model for the evaluation of nutrient bioaccessibility and functionality across time. At the slaughterhouse, one sow intestine was harvested following Maastricht criteria for organ donation after circulatory death (DCD) for transplantation purposes. Duodenum tract was isolated and perfused in sub-normothermic conditions with heterologous blood after cold ischemia induction. Duodenum segment perfusion model was maintained under controlled pressure conditions through extracorporeal circulation for 3 hours. Blood samples from extracorporeal circulation and luminal content samples were collected at regular intervals for the evaluation of glucose concentration by glucometer, minerals (Na+, Ca2+, Mg2+, K+) by ICP-OES, lactate-dehydrogenase and nitrite oxide by spectrophotometric methods. Dacroscopic observation showed peristaltic activity caused by intrinsic nerves. Glycemia decreased over time (from 44.00±1.20 mg/dL to 27.50±0.41; p < 0.01), suggesting glucose utilization by the tissue confirming the organ viability in line with histological examinations. At the end of the experimental period, intestinal mineral concentrations were lower than their level in blood plasma suggesting their bioaccessibility (p < 0.001). A progressive increase of LDH concentration over time was observed in the luminal content probably related to a loss of viability (from 0.32±0.02 to 1.36±0.02 OD; p < 0.05) confirmed by histological findings that revealed a de-epithelization of the distal portion of duodenum. Isolated swine duodenum perfusion model satisfied the criteria for studying bioaccessibility of nutrients, offering a variety of experimental possibilities in line with 3Rs principle.