The effect of initial stages of pulping of spruce, resembling prehydrolysis and alkaline cooking was studied using CP/MAS 13C-NMR, X-ray scattering, FSP and carbohydrate composition in order to study the impact of the pre-treatments on the fiber wall nanostructure. Removal of fiber wall components, hemicellulose and lignin, increased the fiber wall porosity and induced cellulose fibril aggregation. The effect of temperature and pH in the treatment on cellulose fibril aggregate size appears to be secondary. It is the removal of hemicellulose that has a profound effect on the supramolecular structure of the cellulose fiber wall. As the amount of hemicellulose dissolved from wood increases, the fibril aggregate size determined by NMR increases as well, ranging from 16 to 28 nm. Specifically, a good correlation between the amount of glucomannan in the fiber wall and the fibril aggregate size is seen. The lower the amount of glucomannan, the larger the aggregate size. Glucomannan thus seems to prevent aggregation as it acts as a very efficient spacer between fibrils. Elemental fibril size determined by NMR, was quite similar for all samples, ranging from 3.6 to 4.1 nm. By combining measurement methods, a more well-resolved picture of the structural changes occurring during was obtained. © 2021, The Author(s).