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[Display omitted] • Chestnut shells extract induced in-vivo antioxidant effects in rats. • Metabolic profiling by LC-ESI-TQ-Orbitrap-MS was done for chestnut shell. • 52 phenolic compounds and metabolites were identified in blood serum. • Potential mild hypolipidemic and hypoglycemic effects were determined. • Histological analysis revealed no structural changes or toxicity signs. Chestnut (Castanea sativa) shells (CSS) are a source of bioactive compounds with well demonstrated in-vitro antioxidant properties. Nevertheless, no in-vivo studies have already evaluated this effect. This study evaluated the effects of the oral daily administration of an eco-friendly CSS extract (50 and 100 mg/kg per body weight (b.w.)) to rats regarding in-vivo antioxidant activity, glucose and lipids levels, and metabolomic profiling of polyphenols by LC-ESI-LTQ-Orbitrap-MS. The results demonstrated the in-vivo antioxidant properties in the animals liver, kidney and blood serum, as well as protective effects against hemolysis and rising of blood glucose and lipids levels. New insights on metabolomic profiling of polyphenols proved their absorption and further biotransformation by phase I (hydrogenation and hydroxylation) and II reactions (glucuronidation, methylation and sulfation). This is the first study that attempted to validate a novel nutraceutical ingredient extracted from CSS by in-vivo assays, corroborating the outcomes screened by in-vitro assays. [ABSTRACT FROM AUTHOR]