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Five sorghum cultivars (Sorghum bicolor (L.) Moench), from Nigeria, Senegal, Burkina Faso and Italy (two), were characterised by analysing the modulation of cellular functional markers and estimated iron bioavailability; the effect of traditional processing, fermentation and cooking, was also investigated. Glutathione peroxidase (Gpx) activity and total protein content were assayed in human colon-derived DLD-1 cell line exposed to raw and processed samples; iron bioavailability was estimated through the application of an algorithm. The Senegal variety showed promising characteristics: no effects on cell protein and GPx, and a threefold higher percentage of estimated iron bioavailability compared with the other varieties. Fermentation significantly reduced cell proteins and GPx by 26% and 58%, respectively, but improved estimated iron bioavailability from 34 to 77%, whereas cooking only reduced the cellular total protein content. The results indicate that selection of traditional varieties and processing methods can have a significant impact on parameters relevant to sorghum nutritional characteristics.