This study investigated the effect of tissue fixation on myelin water fraction (MWF), an MR derived measurement of myelin content. MWF was found to increase during aldehyde fixation due to an increase in myelin water. Differences in MWF between immersion fixation and perfusion fixation with immersion post-fixation were quantified. This study demonstrated that the measured MWF is sensitive to the changes induced by chemical fixation. The results bridge the interpretation of MWF in the in vivo situation to that of the ex vivo situation and provide a guideline for designing MWF studies with histological validation.