UNLABELLED: High-density lipoprotein (HDL) are capable of losing their physiological functions under pathophysiological conditions and transforming into particles with pro-inflammatory activity. The aim of the study was to evaluate the applicability of dysfunctional changes in HDL as biomarkers of metabolic disorders in hyperlipidemia modeling in vivo. MATERIAL AND METHODS: The experiment was conducted using 70 male Wistar rats with an initial body weight of 234±3 g for 60 days. Disturbances in lipid metabolism in experimental rats were induced by a high-fat diet (50% fat by calorie content) and the addition of 0, 0.5, 1 and 2% cholesterol. A complete blood count and a biochemistry blood serum test were performed. To assess markers of dysfunctional changes in HDL, a fraction of these particles was obtained by sequential ultracentrifugation in KBr solutions of varying density. The relative content of protein, triglycerides, cholesterol was determined in it using the biuret, glycerokinase, and cholesterol oxidase methods, respectively; choline-containing phospholipids were assessed using the phospholipase-choline oxidase colorimetric method on an automatic biochemical analyzer; sphingosine-1-phosphate was determined using the competitive ELISA method; and MDA was evaluated by fluorometric analysis. RESULTS: Hematological parameters indicated the development of anemic syndrome, iron deficiency anemia in particular. In animals of the experimental groups treated with cholesterol, the severity of anemia increased in dose-dependent manner. A decrease in the protein/cholesterol ratio in HDL particles was found in rats treated with high-fat diet with cholesterol, characterizing the acquisition of a dysfunctional phenotype by HDL particles at the background of diet-induced hyperlipidemia (increase in total cholesterol blood serum level). Besides, a significant decrease in triglycerides in HDL particles was observed in the group of rats fed high-fat diet with the highest (2%) quota of cholesterol. A depletion of HDL particles in sphingosine-1-phosphate is shown for this group too, what is also characteristic of dysfunctional HDL. CONCLUSION: The proposed biomarker complex of dysfunctional changes in HDL reflected alterations in lipid metabolism parameters when modeling hyperlipidemia in vivo. At the same time, differences were found in individual indicators of the dysfunctional HDL phenotype in animal (rat) and human models. A decrease in the relative level of triglycerides in HDL particles was found in our experiment, whereas, on the contrary, hyperlipidemia in humans is characterized by HDL enriched in triglycerides. Extrapolation of the obtained experimental data to humans should be carried out taking into account the characteristics of animal species and model used.