Introduction: Plasminogen activator inhibitor-1 (PAI-1) is a marker of vascular endothelial cell activation. The aim of this study was to evaluate whether a difference in low-density lipoprotein cholesterol (LDL-C) concentration affects the PAI-1 level during acute strenuous exercise. Subjects and Methods: Thirteen healthy trained men aged 19 to 23 years participated in this study: 7 of these were categorized in the low LDL-C (LL) group (LDL-C concentration < 100 mg/dL), and 6 in the high LDL-C (HL) group (LDL-C concentration, 100-140 mg/dL). Venous blood samples were collected from the subjects before and after they performed the Cooper 12-min test (running as far as possible within 12 min). LDL-C concentrations were measured by direct methods using samples collected before exercise, and PAI-1 (total amount of active form, latent form, tissue plasminogen activator/PAI-1 complex) level was measured by Latex Photometric Immunoassay using samples collected before and after exercise. LDL-C concentrations in the LL and HL groups were compared using an unpaired t-test. PAI-1 levels were compared using a two-way analysis of variance (group × time) with repeated measures. Statistical significance was set at P < 0.05. Results: The mean (± SE) age, height, and weight of the subjects in the LL group were 21.0 ± 0.5 years, 172.8 ± 1.7 cm, and 64.7 ± 3.0 kg, respectively, and the corresponding values for the subjects in the HL group were 20.6 ± 0.5 years, 171.0 ± 3.5 cm, and 84 ± 7.8 kg, respectively. LDL-C concentration was significantly higher in HL group (121.8 ± 4.4 mg/dL) than in the LL group (87.5 ± 2.8 mg/dL; P < 0.05). There was a significant difference in PAI-1 level between the LL and HL groups (F=0.54; P < 0.05). PAI-1 level before (30.4 ± 6.5 mg/dL) and after (25.2 ± 7.2 mg/dL) exercise did not change significantly in the LL group , whereas it increased significantly in the HL group (40.6 ± 7.0 mg/dL to 58.7 ± 7.8 mg/dL; P < 0.05). There were no significant differences in the PAI-1 levels before exercise in the LL and HL groups, whereas the levels after exercise were significantly different (P < 0.05). Conclusions: It was reported that PAI-1 level does not decrease or change significantly after acute strenuous exercise. In this study, the PAI-1 level increased significantly in the HL group. Therefore, we conclude that a difference in LDL-C concentrations affects vascular endothelial cell activation during acute strenuous exercise.