Eighty-two percent of sexually active women aged 15–44 have used oral contraceptive pills (OCP) in the United States. The OCP, an exogenous source of synthetic forms of steroid hormones, prevents ovulation. Hormone changes during the menstrual cycle (MC) are believed to have an impact on anterior cruciate ligament (ACL) laxity due to estrogen. Because the estrogen receptor β resides on human connective tissue, OCP may have potential impact on tendon and ligament synthesis, structure, and biomechanical properties. Temperature has also been known to have an effect on tissue elasticity. Therefore, the purpose of this study was to investigate the differences in ACL elasticity, force to flex the knee (FFK), and knee flexion–extension hysteresis (KFEH) between OCP users and non-OCP users. To investigate these changes, two different knee temperatures were measured. Nineteen young females were divided into two groups: OCP users and non-OCP users. Blood for estradiol serum concentration (E2) was taken before beginning the tests. ACL elasticity, FFK, and KFEH were assessed both at ambient temperature (22 °C) and after 38 °C warming of the leg to stabilize tissue temperature. Assessments were performed four times during the MC. Throughout the MC, ACL elasticity, FFK, and KFEH fluctuated in non-OCP users, but not in OCP users. At ambient temperature, ACL elasticity was significantly lower and FFK and KFEH were significantly higher in OCP users than non-OCP users (p < 0.05). But, no significant differences in FFK and KFEH between the two groups were found after warming to 38 °C.