AbstractAn investigation into whether the addition of a commonly used anti‐coagulant agent like ethylenediaminetetraacetic acid (EDTA) has an impact on the adhesion potential of blood to non‐porous substrates was conducted. Two non‐porous substrates (aluminum and polypropylene) exhibiting six different surface roughness categories (R1–R6) were used as test substrates upon which either whole blood or blood treated with EDTA was deposited. Samples were exposed to different drying periods (24 hours, 48 hours, and 1 week) before undergoing a tapping agitation experiment in order to evaluate the adhesion to the surface. Clear differences in adhesion potential were observed between whole blood and blood treated with EDTA. Blood treated with EDTA displayed a stronger adhesion strength to aluminum after a drying time of 24 h pre‐agitation, while whole blood presented with a stronger adhesion strength at the drying time of 48 h and 1 week. Both EDTA‐treated and EDTA‐untreated blood was shown to dislodge less easily on polypropylene with the only difference observed on smooth surfaces (0.51–1.50 μm surface roughness). Thus, when conducting transfer studies using smooth hydrophobic substrates like polypropylene or considering the likelihood of transfer given specific case scenarios, differences in adhesion strength of blood due to hydrophobic substrate characteristics and a decreased surface area need to be considered. Overall, whole blood displayed a better adhesion strength to aluminum, emphasizing that indirect transfer probability experiments using EDTA blood on substrates like aluminum should take an increased dislodgment tendency into account in their transfer estimations.