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Introduction Running is a popular recreational activity worldwide, with women’s participation growing rapidly over the past decade. Compared with men, women runners are more likely to sustain a running-related injury, such as bone stress and knee injuries. Following a serious knee injury and subsequent surgery, women also experience worse knee and health-related outcomes than men. However, little is known about the intersection of female-specific health, physical, psychological and social-gendered factors with knee health in women runners with and without a history of knee surgery.Methods and analysis Building on the established ‘TRAjectory of knee heaLth in runners’ ( TRAIL) prospective cohort study and designed with patient and content-expert partners, the nested TRAIL-W study will comprehensively explore the associations of multiple factors (ie, female-specific health, physical, psychological and social gendered) with knee health (symptoms and structural features), device-measured running load and running-related pain in women runners. Where appropriate, we will explore sex and/or gender differences. Alongside their scheduled TRAIL 6-monthly data collection, all active TRAIL female and male participants will be invited to complete a once-off ‘TRAIL-W survey’ and attend an additional laboratory-based assessment. The survey will include questionnaires measuring psychological, social gendered and, for women only, female-specific health factors. The laboratory assessment will measure body composition, bone mineral density and blood biomarkers. A subset of women will be invited to participate in qualitative interviews to understand women runners’ experiences of female-specific health factors and their association with running behaviour.Ethics and dissemination Findings from TRAIL-W, approved by the La Trobe University Human Ethics Committee, will address critical research gaps by describing and exploring the diverse factors that may influence women runners’ knee health.