Collagen is the most prominent protein in biological tissues. Tissue fixation is often required for preservation or sectioning of the tissue. This may affect collagen nanostructure and potentially provide incorrect information when analyzed after fixation. We aimed to unravel the effect of 1) ethanol and formalin fixation and 2) 24 h air-dehydration on the organization and structure of collagen fibers at the nano-scale using small and wide angle X-ray scattering. Samples were divided into 4 groups: ethanol fixed, formalin fixed, and two untreated sample groups. Samples were allowed to air-dehydrate in handmade Kapton pockets during the measurements (24 h) except for one untreated group. Ethanol fixation affected the collagen organization and nanostructure substantially and during 24 h of dehydration dramatic changes were evident. Formalin fixation had minor effects on the collagen organization but after 12 h of air-dehydration the spatial variation increased substantially, not evident in the untreated samples. Generally, collagen shrinkage and loss of alignment was evident in all samples during 24 h of dehydration but the changes were subtle in all groups except the ethanol fixed samples. This study shows that tissue fixation needs to be chosen carefully in order to preserve the features of interest in the tissue.