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New functional foods are increasingly sought to improve the treatment of diseases related to glucose and lipid metabolism. Lupin (Lupinus albus) is an excellent candidate since it exhibits several health-promoting effects. Such beneficial effects can be enhanced by technological treatments like protein hydrolysis with microbial proteases. The aim of this study was to assess the effect of lupin protein hydrolyzate, combined or not with lupin insoluble fiber, on different plasmatic, hepatic, renal and large intestine parameters using an in vivo experimental model of diet-induced hypercholesterolemia. Lupin protein hydrolyzate and insoluble fiber residue were obtained by aqueous protein extraction and sequential hydrolysis with proteases from Bacillus licheniformis and Aspergillus oryzae. The protein hydrolyzate was effective at reducing plasma and hepatic triglycerides, and showed promising effects on glucose metabolism as well as protection against dietary-induced renal alterations. The insoluble fiber residue increased fecal fat excretion, and improved parameters of large intestine physiological status due to its fermentative and water holding capacity.