Edible filamentous fungus or mycoprotein, is promising sustainable protein sources and its protein quality plays a crucial role in their nutritional value. It is hypothesized that prolonged cultivation of filamentous fungus would reduce protein content and increase cell wall thickness, thereby hampering its protein digestibility. Filaementous fungi R. oryzae was cultivated for 24, 48, and 72 hours at two aeration rates (0.3 and 1 vvm) and three pH levels (4, 5.5, and 7). Biomass was analyzed for crude protein content, amino acid composition, cell wall content, and amino acid bioaccessibility using INFOGEST in vitro gastrointestinal digestion model. Prolonged cultivation significantly reduced protein content, while the amino acid profile remained stable across treatments and was notably rich in lysine (8.5–9.4%). In addition, cell wall content increased with cultivation time, accompanied by a reduction in amino acid bioaccessibility.