The high risk of anterior cruciate ligament (ACL) injuries in female athletes may be related to hormonal fluctuations resulting in an increased laxity of ligaments and muscles. This study examined changes in lower limb musculotendinous stiffness (MTS) and knee laxity over the course of the menstrual cycle and investigated the interaction of warm-up on MTS. Eleven female netball players aged 16-18 years who were not using hormonal contraceptives and demonstrated regular menstrual cycles participated in this study. Test-sessions were conducted at onset of menses, mid-follicular phase, ovulation and mid-luteal phase. ACL laxity was determined at each test-session using a KT2000 knee arthrometer. MTS was assessed prior to, and following a standardised warm-up. Repeated measures ANOVA revealed significant (P < 0.05) main effects of test-session and warm-up on MTS. MTS was found to significantly decrease by 4.2% following the warm-up intervention. MTS was significantly lower at week 3 (ovulatory phase) in contrast to weeks 1 and 2 (8.7 and 4.5%, respectively). For knee laxity measures, repeated measures ANOVA revealed no significant (P < 0.05) differences across the menstrual cycle. A reduction in MTS results in greater reliance on reflexive response from the contractile components of the muscle due to a decreased contribution from passive elastic structures and will also increase electromechanical delay. Given that extreme loads are applied to the knee joint within milliseconds, the contractile components cannot respond quickly enough to counteract these sudden and potentially damaging forces. These effects are augmented following a moderate warm-up. Oestrogen fluctuations had no significant effect on anterior knee laxity, however, the effects on MTS over the 28-day cycle were considerable. Future studies should use matched subjects who are using the monophasic oral contraceptive pill to investigate the effects of oestrogen supplementation on lower limb MTS.