This study aimed to develop the first valid questionnaire to assess menstrual cycle (MC) and hormonal contraceptive (HC) knowledge in athletes and sports performance support staff. Questions were developed following a literature review and four knowledge domains were identified: (1) Normal MC Function, (2) MC Dysfunction, (3) Oral Contraceptive Pills, and (4) Other HCs. Experts (n = 6) reviewed the initial questionnaire, followed by pre-testing with athletes and support staff (n = 19). Validity (item analysis, construct validity) and reliability (test-retest, internal consistency) were assessed following questionnaire administration to athletes and staff without MC education ("Low Knowledge" [LK] group; n = 156) and "High Knowledge" [HK] participants (n = 30) with MC education. Most (n = 122) LK participants completed the questionnaire twice to assess test-retest reliability. Expert review showed good agreement (> 80%) on item clarity and relevancy and pre-testing indicated good content and face validity. The HK group scored significantly higher than LK participants across the questionnaire (43 ± 5 vs. 28 ± 10; p < 0.001; d = 1.48 [1.05, 1.90]) and all knowledge domains (p < 0.05). High item discrimination parameters were found for all domains, indicating effective discrimination between respondents with different knowledge levels. Test-retest reliability was excellent (intra-class correlation coefficient estimates from 0.93 [0.90, 0.95] to 0.98 [0.96, 0.99]), with all domains showing good-to-excellent reliability. Internal reliability (Cronbach's alpha) was considered acceptable (0.93 [0.92, 0.95]; a >= 0.7). This questionnaire delivers a valid and reliable tool to assess MC and HC knowledge amongst athletes and support staff and is recommended to guide and evaluate educational interventions to improve menstrual health literacy within sport.
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