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This study assessed the influence of estrogen (E2) on muscle damage biomarkers [skeletal muscle-creatine kinase (CK); cardiac muscle-CK-MB] responses to prolonged aerobic exercise. Eumenorrheic women (n= 10) who were physically active completed two 60-minute treadmill running sessions at~ 60-65% maximal intensity during low E2 (midfollicular menstrual phase) and high E2 (midluteal menstrual phase) hormonal conditions. Blood samples were collected prior to exercise (following supine rest), immediately post-, 30 min post-, and 24 hours post-exercise to determine changes in muscle biomarkers. Resting blood samples confirmed appropriate E2 hormonal levels Total CK concentrations increased following exercise and at 24 hours postexercise were higher in the midfollicular low E2 phase (p< 0.001). However, CK-MB concentrations were unaffected by E2 level or exercise (p= 0.442) resulting in the ratio of CK-MB to total CK being consistently low in subject responses (ie, indicative of skeletal muscle damage). Elevated E2 levels reduce the CK responses of skeletal muscle, but had no effect on CK-MB responses following prolonged aerobic exercise. These findings support earlier work showing elevated E2 is protective of skeletal muscle from exercise-induced damage associated with prolonged aerobic exercise.