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Site-specific nitrogen (N) management in precision agriculture is used to improve nitrogen use efficiency (NUE) at the field scale. The objective of this study has been (i) to better understand the relationship between data derived from an unmanned aerial vehicle (UAV) platform and the crop temporal and spatial variability in small fields of about 2 ha, and (ii) to increase knowledge on how such data can support variable application of N fertilizer in winter wheat (Triticum aestivum). Multi-spectral images acquired with a commercially available UAV platform and soil available mineral N content (Nmin) sampled in the field were used to evaluate the in-field variability of the N-status of the crop. A plot-based field experiment was designed to compare uniform standard rate (ST) to variable rate (VR) N application. Non-fertilized (NF) and N-rich (NR) plots were placed as positive and negative N-status references and were used to calculate various indicators related to NUE. The crop was monitored throughout the season to support three split fertilizations. The data of two growing seasons (2017/2018 and 2018/2019) were used to validate the sensitivity of spectral vegetation indices (SVI) suitable for the sensor used in relation to biomass and N-status traits. Grain yield was mostly in the expected range and inconsistently higher in VR compared to ST. In contrast, N fertilizer application was reduced in the VR treatments between 5 and 40% depending on the field heterogeneity. The study showed that the methods used provided a good base to implement variable rate fertilizer application in small to medium scale agricultural systems. In the majority of the case studies, NUE was improved around 10% by redistributing and reducing the amount of N fertilizer applied. However, the prediction of the N-mineralisation in the soil and related N-uptake by the plants remains to be better understood to further optimize in-season N-fertilization.