In a first experiment, the effect of in vitro incubation with Aspergillus niger (An) on the chemical composition of different fibrous substrates was studied. In a second experiment, the effect of incubation time (0,72 and 144 h) on in vitro digestion with An of dry matter (IVDMD), neutral detergent fiber (DVNDF) and acid detergent fiber (DVADF) was evaluated for different substrates. Wheat straw, WS; barley straw, BS; oat hulls, OH; dehydrated alfalfa, DA; and dehydrated ryegrass, DB substrates were evaluated. In both experiments, incubations were performed in 250 mL Erlenmeyer flasks; 2 g of substrate was added to a culture medium (pH = 6), and incubated at 28 °C with constant ventilation. The An dose consisted of 3 mL of a solution of 5.3 χ 10(6) spores mL-1 per flask. Incubation time for the first experiment was 144 h. After incubation, the contents of the flasks were homogenized in a blender for one minute. Subsequently, bromatological analysis was conducted without separating the An biomass. No interaction effects among any studied variables were observed. An incubation effected cell wall (NDF) and lignocellulose (ADF) content. An increase in true protein (TP) and soluble nitrogen (SN) was observed for all substrates tested, but responses differed depending on the type of substrate. The largest increases in TP were observed in DA and DB substrates (0.55 and 0.63% DM, respectively). IVDMD, DVNDF and DVADF were affected by the type of substrate and incubation time. The highest value of IVDMD144 was observed in the DAAn treatment (49.47% DM), followed by the DBAn treatment, with a value of 45.51% DM. OHAn showed the lowest value of IVDMD144 (29.38% DM). The results suggest that An possesses fibrolytic and metabolic potential for improving the nutritional value of fibrous foods through of the digestion of structural carbohydrates and the liberation of nitrogenous fractions embedded in cell walls, producing a change in the chemical composition and the potential digestibility of treated foods.