Scope Bilberry, bananas, and apples are used for smoothie production because the health-promoting activities and to prevent human diseases including neurodegenerative disorders. The smoothie is prepared to promote a promising practice for increasing the intake of fruit in the diet. Methods and results The smoothie is packed into dark glass jars, pasteurized, and stored for up to 4 months at 4 or 22 °C. Then, it is analyzed for the polyphenols profile using liquid chromatography-high resolution mass spectometry (LC-HRMS) Polyphenols content and the antiinflammatory, anticholinesterase, and antioxidant activities, and the impact on catalase activity are controlled using biochemical analyses. A significant decrease in the flavanol content (p < 0.05) is investigated, while there are lower decreases or no changes in the other polyphenols content in the smoothies stored at 4 °C. The changes in the anticholinesterase and antioxidant activities of the smoothie are correlated with the total polyphenols, anthocyanins, flavonols, and tannins content. Conclusion The proposed preservation of the smoothie and its storage at refrigeration temperature is adequate to maintain the smoothie's nutritional and functional effect for a 4-month shelf life. Even significant changes in the content of individual subgroups of polyphenols are not drastically reflected in the decrease of the smoothie biological activities.