The purpose of this study was 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 behavior of the Achilles tendon in vivo. Twenty women (age 28.0 +/- 4.2 yr, height 1.67 +/- 0.07 m, mass 61.6 +/- 6.8 kg) who had been using the MOCP for at least 12 mo together with 20 matched women who were non-MOCP users (age 31.9 +/- 7.3 yr, height 1.63 +/- 0.05 m, mass 62.5 +/- 5.9 kg) participated in this study. Non-MOCP users were tested at the time of lowest (menstruation) and highest (approximately same as ovulation) estrogen, whereas 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 while 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. Repeated-measures ANOVA revealed a significant (P < 0.05) main effect of subject group with significantly lower Achilles strain (25.5%) in the MOCP users compared with the non-MOCP users. In conclusion, acute fluctuations in plasma estrogen across the menstrual cycle in non-MOCP users did not alter the strain behavior 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.