Background: Several studies have shown an association between abacavir (ABC) and increased risk of myocardial infarction (MI), but the causative mechanism has not been established. Both vascular endothelial inflammation and platelet activation have been proposed as contributing factors. Objective: The study aims to investigate the effects of ABC relative to tenofovir disoproxil (TDF) on functional assays of primary and secondary hemostasis and a comprehensible range of relevant biomarkers. Methods: In an investigator-initiated, open-labeled, crossover trial, we included HIV-infected males receiving either ABC or TDF and switched treatment to the alternate drug. At inclusion and after three months on the new regimen, we performed Multiplate® and thromboelastography (TEG® ) and measured biomarkers of coagulation, inflammation, platelet reactivity, endothelial disruption and activation, and fibrinolysis, lipids, HIV RNA, CD4, CD8, and creatinine. Treatment effects were assessed by comparing intraindividual differences between the two treatment orders by the Wilcoxon Rank Sum test. Results: In total, 43 individuals completed the study. No intraindividual differences were observed for Multiplate® or TEG® when switching between regimens. We observed a significant treatment effect on coagulation factors II-VII-X (p<0.0001), sCD40L (a biomarker of platelet reactivity, p=0.04), thrombomodulin (biomarker of endothelial damage, p=0.04), lipids, and CD8 cell counts (p=0.04), with higher values during ABC treatment compared to TDF. Conclusion: Compared to TDF, ABC treatment affected several outcome measures in a pro-coagulant direction. Suggesting that the risk of MI associated with ABC may be caused by the sum of multiple, discrete disturbances in the hemostatic system and endothelium. Study Registration: The trial was registered at clinicaltrials.gov (NCT02093585).