Hardness and elastic modulus of spruce wood cell walls parallel to their axial direction were investigated by means of nanoindentation. In the secondary cell wall layer S2 of individual earlywood and compression wood tracheids, a systematic pattern variability was found. Several factors potentially affecting nanoindentation results were investigated, i.e. specimen orientation related to the indenter tip, cutting direction during specimen preparation, tip geometry, specimen and fibre inclination, respectively, and finally micro fibril orientation. Mechanical property measurements were correlated with structural features measured by confocal Raman spectroscopy. It was demonstrated that very high variability in the measurement of micromechanical cell wall properties can be caused by unintentional small fibre misalignment by few degrees with regard to the indentation direction caused by sub-optimal specimen preparation.