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Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm., is one of the most widely consumed mushrooms in the world with interesting health-promoting benefits, mainly due to its richness in several bioactive compounds [1]. Mushrooms produce ergosterol as one of their main sterols, which has been considered a contributor to their anti-inflammatory, antioxidant, and antitumor properties [2]. Obtaining an ergosterol enriched extract depends on different variables, such as the extraction method, solvent type, temperature, extraction time, and the solid-liquid ratio [3]. Therefore, it is essential to define the main variables and relevant response criteria to maximize the extraction yield and purity, combining the economic competitivity. In the present work, response surface methodology (RSM) was applied to optimize a heat assisted extraction system (HAE), combining time (t) and temperature (T) effects, and using a circumscribed central composite design (CCCD) for the recovery of ergosterol from the fruiting bodies of P. ostreatus produced with lignocellulose substract. The obtained responses were the quantification of ergosterol by HPLC-UV (Y1: mg of ergosterol per g of extract residue and Y2: mg of ergosterol per 100 g of dry weight mushroom), and the extraction yield (Y3: %). The CCCD consist of 16 response combinations and 4 centre points. Response surface models were fitted by using the following second order polynomial equation. The results obtained showed a significant interaction between the variables. For all the three responses (Y1, Y2, and Y3), the model successfully explained more than 80% variation in the experimental data (i.e. R2˃8). The …