Exhaustive exercise has been associated with an increased risk for URTI in mice and humans. We have previously shown that female mice are better protected from the lethal effects of HSV-1 infection, both at rest and following exercise stress, but little is known about possible mechanisms. This study tested the effects of estrogen on HSV-1 infection and macrophage antiviral resistance following repeated exhaustive exercise. Female mice were assigned to either exercise (Ex) or control (C): intact female (I-C or I-Ex), ovariectomized female (O-C or O-Ex), or ovariectomized estrogen-supplemented female (E-C or E-Ex). Exercise consisted of treadmill running to volitional fatigue (~125 min) for 3 consecutive days. Intact female mice had a later time to death than O and E (p < 0.05) and fewer deaths than both O and E (p < 0.05). Exercise stress was associated with increased time to sickness (p < 0.05) and symptom severity at days 6 and 12-21 post-infection (p < 0.05) and decreased macrophage antiviral resistance (p < 0.001) in all groups. E had increased symptom severity at days 6 and 13-21 post-infection (p < 0.05). Results indicate that intact female mice are better protected from the lethal effects of HSV-1 infection and that exercise stress had a similar negative impact in all groups. This protective effect was lost in ovariectomized mice, but was not reinstated by 17-estradiol replacement. This indicates that other ovarian factors, alone or in combination with estrogen, are responsible for the protective effects in females.