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, lupin seeds have emerged as a novel and valuable source of proteins representing a sustainable alternative to current raw materials of plant-based proteins, such as soybean. In this regard, lupin demonstrates superior adaptation to the Mediterranean climate with improved tolerance to water stress. However, its suitability for human consumption is limited due to the presence of anti-nutritional and anti-technological factors, including alkaloids, polyphenols, tannins, and lipids, which can have adverse nutritional consequences and/or impact on the purity and yield of protein extraction. In this study, the Lupinus luteus seeds were germinated for 1, 2, 3, and 6 days and the effect of germination on the anti-nutritional and anti-technological factors of flours and derived protein isolates was analyzed. Additionally, changes on techno-functional properties of lupin protein isolates were also studied. Results showed that prolonged germination decreased fat content whereas antioxidant activity, polyphenols, saponin, and alkaloid content of flours increased. However, alkaloids were completely removed during protein extraction. Furthermore, protein isolates derived from germinated seeds exhibited higher water and oil absorption capacities, as well as improved foaming and emulsifying capabilities in comparison to isolates from non-germinated seeds. Nevertheless, prolonged germination periods were associated with diminished foam and emulsion stability. Therefore, germination of lupin seeds for no longer than 3 days should be recommended in order to minimize the levels of anti-nutritional and anti-technological factors, while enhancing techno-functional properties of the isolate.