Eight dual flow continuous culture fermenters were used in three experimental periods to evaluate effects of diets formulated to contain different amounts of rumen undegradable protein on microbial fermentation and nutrient flow. Two dietary treatments contained 51% forage (28.5% alfalfa haylage and 22.6% maize silage) and 49% concentrate on a DM basis. The concentrate mix consisted of 28.9% ground maize, 15.9% soyabean meal (SBM) and 2.0% animal fat for the control diet (CTRL), or 30.4% ground maize, 2.3% SBM, 9.0% expeller processed SBM (ES), 3.1% fish meal (FM) and 2.0% animal fat for the low degradable protein diet (ESFM). Dietary energy (1.74 Mcal kg-1), crude protein (CP, 19.2%) neutral detergent fiber (NDF, 28.8%) and acid detergent fiber (ADF, 18.6%) content did not differ between diets. Control and ESFM diets were formulated to contain 68% or 55% rumen degradable protein, respectively. True OM and DM digestion were not affected (P > 0.05) by dietary treatment. Digestion of NDF (43.1 versus 38.8%, P = 0.07) and ADF (59.3 versus 54.7%, P < 0.05) was lower when fermenters received ESFM rather than CTRL diet. Although total VFA concentration was not affected (P > 0.05) by dietary treatment, molar proportion (mol per 100 mol) of propionate (29.2 versus 24.2, P < 0.05) was higher, and acetate (48.8 versus 53.8, P < 0.05) and acetate to propionate ratio (2.25 versus 1.70, P < 0.05) lower for the ESFM compared with CTRL. Changes in VFA fermentation patterns were possibly due to fat composition of the protein supplements. Ammonia nitrogen concentration in the effluent, and non-ammonia, dietary and bacterial nitrogen flow from the fermenters were not affected (P > 0.05) by dietary treatment. Efficiency of bacterial protein synthesis and dietary crude protein degradation were not affected (P > 0.05) by dietary treatment. Partial replacement of SBM by ES and FM decreased fiber digestion and altered VFA molar proportion in the effluent, but did not reduce ruminal degradation of dietary protein.