The analysis of forensic footwear evidence often requires the preparation of test impressions created under controlled laboratory conditions. When these test impressions are compared to questioned impressions, (dis)agreement in physical size is an important attribute that must be evaluated and documented. Integral to this comparison is an understanding of the variation that may exist between replicate test impressions, and test impressions created using different methods. The aim of this study was to empirically characterize the variation that exists within and between test impressions prepared using a static benchtop and a dynamic walking method, as well as explore the potential influence of the wearer’s foot size when using the walking method. To examine this variation, twenty-three participants were recruited to prepare test impressions of two different shoe makes and models in four different manufacturer’s sizes. Five replicate benchtop impressions per make/model/size and three replicate walking impressions per participant/make/model/size were created, resulting in approximately 550 test samples, to which an additional 150 quality control copies were blindly added, resulting in almost 700 processed test impressions. Using reproducible and reliable ground control points, the physical size of toe-to-heel length and medial-to-lateral ball of the toe width measurements were collected and compared. For the shoe make/models examined in this study, a systematic bias was observed between benchtop and walking impressions, such that benchtop impressions were almost always longer and narrower than walking impressions, and that within the walking method, physical size differences vary with foot/shoe size mismatch. Of the experimental shoes and groups examined in this study, the variation in toe-to-heel length measurements between benchtop and walking impressions was greatest when the shoe was two sizes smaller than the foot, resulting in a maximum physical size difference of 4.18 mm for a Nike® Downshifter 11 outsole. Similarly, when comparing the variation in toe-to-heel length measurements between walking impressions created using a correctly-fitted shoe versus a shoe that was two sizes smaller than the wearer’s foot, a maximum physical size difference of 3.25 mm was observed. Based on these findings, best practice suggests that footwear analysts document the foot size of the wearer preparing walking test impressions, refrain from using benchtop impressions to form opinions about physical size consistency/differences, and to be cognizant that a large mismatch between shoe/foot size when creating walking impressions can lead to length differences greater than 3.0 mm.