Menstrual cycle tracking apps (MCTAs) have potential in epidemiological studies of women's health, facilitating real-time tracking of bleeding days and menstrual-associated signs and symptoms. However, information regarding the characteristics of MCTA users versus cycle nontrackers is limited, which may inform generalizability. We compared characteristics among individuals using MCTAs (app users), individuals who do not track their cycles (nontrackers), and those who used other forms of menstrual tracking (other trackers). The Ovulation and Menstruation Health Pilot Study tested the feasibility of a digitally enabled evaluation of menstrual health. Recruitment occurred between September 2017 and March 2018. Menstrual cycle tracking behavior, demographic, and general and reproductive health history data were collected from eligible individuals (females aged 18-45 years, comfortable communicating in English). Menstrual cycle tracking behavior was categorized in 3 ways: menstrual cycle tracking via app usage, that via other methods, and nontracking. Demographic factors, health conditions, and menstrual cycle characteristics were compared across the menstrual tracking method (app users vs nontrackers, app users vs other trackers, and other trackers vs nontrackers) were assessed using chi-square or Fisher exact tests. In total, 263 participants met the eligibility criteria and completed the digital survey. Most of the cohort (n=191, 72.6%) was 18-29 years old, predominantly White (n=170, 64.6%), had attained 4 years of college education or higher (n= 209, 79.5%), and had a household income below US $50,000 (n=123, 46.8%). Among all participants, 103 (39%) were MCTA users (app users), 97 (37%) did not engage in any tracking (nontrackers), and 63 (24%) used other forms of tracking (other trackers). Across all groups, no meaningful differences existed in race and ethnicity, household income, and education level. The proportion of ever-use of hormonal contraceptives was lower (n=74, 71.8% vs n=87, 90%, P=.001), lifetime smoking status was lower (n=6, 6% vs n=15, 17%, P=.04), and diagnosis rate of gastrointestinal reflux disease (GERD) was higher (n=25, 24.3% vs n=12, 12.4%, P=.04) in app users than in nontrackers. The proportions of hormonal contraceptives ever used and lifetime smoking status were both lower (n=74, 71.8% vs n=56, 88.9%, P=.01; n=6, 6% vs n=11, 17.5%, P=.02) in app users than in other trackers. Other trackers had lower proportions of ever-use of hormonal contraceptives (n=130, 78.3% vs n=87, 89.7%, P=.02) and higher diagnostic rates of heartburn or GERD (n=39, 23.5% vs n=12, 12.4%, P.03) and anxiety or panic disorder (n=64, 38.6% vs n=25, 25.8%, P=.04) than nontrackers. Menstrual cycle characteristics did not differ across all groups. Our results suggest that app users, other trackers, and nontrackers are largely comparable in demographic and menstrual cycle characteristics. Future studies should determine reasons for tracking and tracking-related behaviors to further understand whether individuals who use MCTAs are comparable to nontrackers.