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Long-term experiments are an important tool to investigate the effects of crop rotation and fertilization measures under climate change conditions. However, there are few studies on nitrogen use efficiency (NUE) of crops in long-term cropping systems in Europe, but no consolidate studies in Germany. We analyzed a long-term experiment established in 1982 to observe the role of legumes on total nitrogen uptake, NUE, agronomic efficiency, nitrogen response index and N recovery efficiency compared to fallow land and cereals in a crop rotation over the long-term. The experiment included five different precrops (fallow land, maize, oat, field bean and clover mulch) in combination with four levels of fertilization (control, PK, PK+N with reduced dosage, and PK+N with full dosage followed by three years of cereal cultivation (winter wheat, winter rye, summer barley) in all treatments. The field trial was conducted as a fully factorial split plot design, with spatially randomized field repetitions in four blocks. Long-term mineral NPK fertilization improved the N uptake of winter wheat (185.7 kg N ha−1), winter rye (152.2 kg N ha−1) and summer barley (92.2 kg N ha−1) when the higher level of fertilization (NPK 100%) was implemented. A year-round application of clover mulch as green manure in the rotational system resulted in higher NUE (up to 37%) compared to fallow land after 40 years. Crop rotation with oat and maize (100% cereals) led to lower NUE (up to 30%) compared to fallow land. Clover mulch (198%), field bean (155%), fallow land (148%) and maize (119%) significantly increased the N uptake of wheat compared to oat in 2015. Over time, the agronomic efficiency was increased. This increase was 4 or 5 times higher in the 9th and 10th crop rotation compared to the beginning of the experiment (in 1983). Nitrogen response index of clover mulch was approximately 33–37% lower than fallow land at the beginning of the experiment in 1983 and 45–50% lower over the 40 years. N recovery efficiency values of winter wheat (94%), winter rye (66%) and summer barley (46%) increased within the 10th crop rotation compared to the beginning of the long-term experiment. Combining clover mulch as a green manure with mineral fertilizer has greater potential to improve NUE and crop productivity by increasing soil N availability. Our study highlights the importance of evaluating agronomic management practices in long-term field experiments to understand their sustainable effects. • Soil nutrition is regulated by the quality & quantity of plant residues incorporated into the soil. • Long-term application of green mulch and mineral fertilizer improves NUE and crop productivity. • After 40 years, agronomic efficiency was about 4 or 5 times higher than at the beginning of the experiment in 1983. • Nitrogen response index of clover mulch was about 33–37% lower than fallow at onset of the experiment. [ABSTRACT FROM AUTHOR]