Purpose: To elucidate the effect of normal fluctuating (non-monophasic oral contraceptive pill (MOCP) users) and low, consistent (MOCP users) endogenous plasma estrogen levels on the strain behaviour of the Achilles tendon in vivo. Methods: Twenty females (Age=28.0±4.2yr; Ht=1.67±0.07m; Mass=61.6±6.8kg) who had been using the monophasic oral contraceptive pill (MOCP) for at least 12 months together with 20 matched, female, non-MOCP users (Age=31.9±7.3yr; Ht=1.63±0.05m; Mass=62.5±5.9kg) participated in this study. Non-MOCP users were tested at the time of lowest (menstruation) and highest (&#8776;ovulation) estrogen whilst MOCP users, who exhibited constant and attenuated endogenous estrogen levels, were tested at Day 1 and Day 14 of their cycle. At each test session, maximal isometric plantarflexion efforts were performed on a calf-raise apparatus whilst synchronous real-time ultrasonography of the triceps surae aponeurosis was recorded. Achilles tendon strain (%) was calculated by dividing tendon displacement during plantarflexion by resting tendon length. Results: Repeated measures ANOVA revealed a significant (p < 0.05) main effect of subject group with significantly lower Achilles strain rates (25.5%) in the MOCP users compared to the non-MOCP users. Conclusions: Acute fluctuations in plasma estrogen across the menstrual cycle in non-MOCP users did not alter the strain behaviour of the Achilles tendon. Conversely, long-term exposure to attenuated estrogen in MOCP users resulted in a decrease in Achilles tendon strain which is thought to be attributed to the effects of endogenous estrogen on collagen synthesis. These findings have a number of important functional and clinical implications.