The initial STACKENOX (STACK-on to ENOXaparin) study investigated the effect of stacking unfractionated heparin (UFH) onto a chronic treatment with enoxaparin, a common practice in interventional cardiology when a patient treated with enoxaparin receives an additional bolus of UFH at the time of percutaneous coronary intervention. The study brought to light some unexpected consequences on coagulation tests and hemorrhagic risk. This substudy was performed to provide a pharmacological explanation for these observations. Seventy-two healthy individuals previously treated with enoxaparin for 2.5 days received a stack-on of 70 IU/kg intravenous UFH dose 4, 6, or 10 h after the last enoxaparin dose. Anticoagulation activity was monitored by activated partial thromboplastin time, anti-Xa and anti-IIa activities and a thrombin generation test. In parallel, plasma samples of the individuals receiving the chronic enoxaparin treatment obtained at 4, 6, or 10 h after the last enoxaparin dose were spiked in vitro with a dose of UFH reproducing the concentration achieved in vivo after the bolus of UFH alone. In-vivo stack-on of UFH induced an over-anticoagulation identified by changes in anti-Xa and, less markedly, in anti-IIa and activated partial thromboplastin time levels, whereas in-vitro UFH spiking, only produced an additive effect. We hypothesize that the potentiation of UFH on anti-Xa activity observed in vivo may caused by the UFH bolus mobilizing enoxaparin chains stored in the endothelial glycocalyx during chronic pretreatment. This over-anticoagulation and its potential hemorrhagic risk after stack-on of UFH have obvious clinical implications.