Purpose: To examine the relationship between menstrual cycle (MC) phase-dependent fluctuations of estrogen and progesterone and virtual cycling race performance, with a secondary aim of correlating perceived MC-related symptoms with performance.Methods: In a novel observational study design, thirty-seven female cyclists/triathletes not using any hormonal contraception completed one virtual cycling race [19.5 km time trial (TT)] per week across a one-month period (totaling four races). Participants completed MC characterization and tracking, including urinary ovulation kits, across two complete MCs. Venous blood samples were collected within 21 h of racing to determine serum 17-β-estradiol and progesterone concentrations, as well as an assessment of self-reported, perceived race-day MC and gastrointestinal (GI) symptoms, which were all then correlated to race performance.Results: There was no relationship between race completion time and individual estradiol (r = -0.001, p = 0.992) or progesterone (r = -0.023, p = 0.833) concentrations. There was no difference between race time between MC phases (follicular/luteal, p = 0.238), whether MC bleeding or not bleeding (p = 0.619) and ovulating or not ovulating (p = 0.423). The total number of perceived MC symptoms recorded on race day was positively correlated to increased race time [r = 0.268 (95% CI 0.056 to 0.457), p = 0.014], as was the number of GI symptoms of at least "moderate" severity before the race (r = 0.233 [95% CI 0.021 to 0.425], p = 0.031), but not post-race (r = 0.022, p = 0.841).Conclusions: When implementing a novel, virtual cycling race, fluctuations in ovarian hormone concentrations across the MC do not appear to affect real-world cycling performance among trained cyclists, while perceived negative MC and GI symptoms may relate to impaired performance. Therefore, the management of negative MC and GI symptoms appears important for athletic performance enhancement or to mitigate performance decline.