Background: The increase in exercise levels in the last few years among professional and recreational female athletes has led to an increased scientific interest about sports health and performance in the female athlete population. The purpose of the IronFEMME Study described in this protocol article is to determine the influence of different hormonal profiles on iron metabolism in response to endurance exercise, and the main markers of muscle damage in response to resistance exercise; both in eumenorrheic, oral contraceptive (OC) users and postmenopausal well-trained women. Methods: This project is an observational controlled randomized counterbalanced study. One hundered and four (104) active and healthy women were selected to participate in the IronFEMME Study, 57 of which were eumenorrheic, 31 OC users and 16 postmenopausal. The project consisted of two sections carried out at the same time: iron metabolism (study I) and muscle damage (study II). For the study I, the exercise protocol consisted of an interval running test (eight bouts of 3 min at 85% of the maximal aerobic speed), whereas the study II protocol was an eccentric-based resistance exercise protocol (10 sets of 10 repetitions of plate-loaded barbell parallel back squats at 60% of their one repetition maximum (1RM) with 2 min of recovery between sets). In both studies, eumenorrheic participants were evaluated at three specific moments of the menstrual cycle: early-follicular phase, late-follicular phase and mid-luteal phase; OC users performed the trial at two moments: withdrawal phase and active pill phase. Lastly, postmenopausal women were only tested once, since their hormonal status does not fluctuate. The three-step method was used to verify the menstrual cycle phase: calendar counting, blood test confirmation, and urine-based ovulation kits. Blood samples were obtained to measure sex hormones, iron metabolism parameters, and muscle damage related markers. Discussion: IronFEMME Study has been designed to increase the knowledge regarding the influence of sex hormones on some aspects of the exercise-related female physiology. Iron metabolism and exercise-induced muscle damage will be studied considering the different reproductive status present throughout well-trained females’ lifespan.