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The fermentation of Triticum dicoccum with sourdough enhances the nutritional aspects of the final product by the enrichment of several compounds with potential medical and biological activity, hence, could improve the health of consumers. This study analyzed the chemical composition of fermented spelt flour from Garfagnana (Province of Lucca, Tuscany) by 1 H Nuclear Magnetic Resonance ( 1 H NMR) spectroscopy and its in vitro antioxidant properties by FRAP and DPPH tests. Beyond this, the ex vivo CAA-RBC assay determined the cellular antioxidant activity on human erythrocytes under oxidative condition. Carbohydrate reduction was observed, while amino acids, organic acids and aromatic compounds with potential antioxidant activity increased during the fermentation time. Moreover, both in vitro and ex vivo outcomes showed an improved antioxidant profile. As a possible industrial application, the fermentation process adopted in this research could be reproduced on a large scale for the commercialization of the products by the food industry., (Copyright © 2019 Elsevier Ltd. All rights reserved.)