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Forest biochemical and biophysical variables and their spatial and temporal distribution are essential inputs to process-orientated ecosystem models. To provide this information, imaging spectroscopy appears to be a promising tool. In this context, the present study investigates the potential of spectral unmixing to derive sub-pixel crown component fractions in a temperate deciduous forest ecosystem. However, the high proportion of foliage in this complex vegetation structure leads to the problem of saturation effects, when applying broadband vegetation indices. This study illustrates that multiple endmember spectral mixture analysis (MESMA) can contribute to overcoming this challenge. Reference OPEN ACCESSRemote Sens. 2015, 7 15362 fractional abundances, as well as spectral measurements of the canopy components, could be precisely determined from a crane measurement platform situated in a deciduous forest in North-East Germany. In contrast to most other studies, which only use leaf and soil endmembers, this experimental setup allowed for the inclusion of a bark endmember for the unmixing of components within the canopy. This study demonstrates that the inclusion of additional endmembers markedly improves the accuracy. A mean absolute error of 7.9% could be achieved for the fractional occurrence of the leaf endmember and 5.9% for the bark endmember. In order to evaluate the results of this field-based study for airborne and satellite-based remote sensing applications, a transfer to Airborne Imaging Spectrometer for Applications (AISA) and simulated Environmental Mapping and Analysis Program (EnMAP) and Sentinel-2 imagery was carried out. All sensors were capable of unmixing crown components with a mean absolute error ranging between 3% and 21%.