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Background The intestinal microbiota is involved in pathogenesis of systemic inflammation in chronic heart failure (HF). The results of several previous studies suggest the need to evaluate the effects of prebiotics in HF. The current study aimed to evaluate the dietary addition of a prebiotic complex on dynamic changes of the mucosa‐associated intestinal microbiota and endotoxemia in rats with experimental HF. Methods The study was conducted on 30 adult female rats in accordance with the “Principles of Laboratory Animal Care”. The animals were divided in 3 groups: 1) control (n=10), 2) HF only (n=10) and 3) HF with addition of a prebiotic complex (HF‐P; n=10). Experimental HF was induced by daily subcutaneous injection of phenylephrine (5 mg/kg/day) followed by swimming exercise until exhaustion for 14 days. In HF‐P group, the animal diet was supplemented with a prebiotic complex, containing wheat bran fermented with Saccharomyces cerevisiae culture (“Eubikor”, St. Petersburg, Russia), starting 7 days before the induction of HF and throughout the study, in a concentration of 2.5 g/kg of body weight. At the end of experimental period, the blood samples were collected from the hearts. The plasma concentration of the endotoxin (lipopolysaccharides [LPS]; nmol/ml) and the microbial markers (higher fatty acids, aldehydes and sterols) with subsequent calculation of the microbial numbers within the intestinal mucous layer (cells/gram×105 ) were determined using the mass spectrometry method. Statistical data analysis was carried out with the PAST 3 software package. Results All animals in experimental groups demonstrated clinical symptoms of HF that was later confirmed by histological examination. The rats in HF group demonstrated significant increase in the concentration of LPS (p = 0.025) and the number of gram‐negative Prevotella spp. (p = 0.015), Fusobacterium spp. (p = 0.008), Kingella spp. (p = 0.06); gram‐positive Enterococcus spp. (p = 0.025), Clostridium propionicum (p = 0.008), Lactobacillus spp. (p = 0.015) bacteria. Furthermore, the number of gut commensal bacteria, such as Bifidobacterium spp., Propionibacterium freudenreichii and Eubacterium spp., was tended to be decreased (p <0.1). The use of a prebiotic complex in HF rats led to a significant increase in the number of the commensal flora, particularly Bifidobacterium spp. (p = 0.025), Propionibacterium freudenreichii. (p = 0.01) and Eubacterium spp. (p = 0.01). Moreover, the animals in HF‐P group revealed a significant decrease in the plasma level of LPS (p = 0.016) and the number of Prevotella spp. (p = 0.01), Fusobacterium spp. (p = 0.005), Helicobacter pylori (p = 0.025), Enterococcus spp. (p = 0.01), Actinomyces viscosus (p = 0.025), Lactobacillus spp. (p = 0.037) bacteria and microscopic fungi producing campesterol and sitosterol (p = 0.016). Conclusion The findings in our study demonstrate that the addition of a prebiotic complex to the diet of HF rats affects the dynamic changes of the intestinal microbiota and decreases the plasma endotoxin leve...