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Perennial grasses can provide very high biomass yields and a better environmental profile compared to most annual cereal crops. The grass biomass can be processed into a protein-rich fraction for monogastric feed and a fibrous pulp fraction for ruminant feed or energy production. We studied the effect of fertilization of tall fescue grown on two sandy sites in Denmark. Dry matter (DM) and crude protein (CP) yield of first cut was measured at four nitrogen (N) fertilization levels (0, 70, 140 or 210 kg N ha−1). Samples were processed in a screw press, achieving a pulp fraction and a juice fraction from which proteins were precipitated into a protein concentrate fraction. CP allocation between the fractions of protein concentrate and pulp was analysed as well as the amino acid (AA) profile. CP in the brown juice fraction was not analysed. CP concentration and CP yield of unprocessed tall fescue increased linearly with N fertilization, attaining concentrations up to 126 g kg−1 DM and yields up to 0.76-0.86 Mg ha−1. N fertilization did not affect CP allocation between concentrate and pulp in a consistent way. As a mean of all treatments and both sites, 17% of CP was recovered in the concentrate and 74% remained in the pulp. CP concentration increased linearly with fertilization in both concentrate and pulp with coefficients of 0.50 and 0.21 g kg−1 DM kg−1 N ha−1, i.e. with a higher increase in the concentrate fraction. The AA profile only differed slightly between the two fractions and between the fertilization levels, however, the total sulphur to N ratio in both fractions decreased with increased fertilization, and a similar trend was observed for the relative content of the sulphur-containing amino acids. The AA profile deviated considerably from soybean meal, particularly with higher leucine and lower glutamic acid content. In conclusion, fertilization can greatly increase CP concentration and CP yield. However, the separation process needs improvements to increase the protein in the concentrate fraction.