Clothing and footwear designed for trail running shed microplastics (MPs) during use. Trail running events may therefore present a significant source of MP pollution in conservation and wilderness areas. Microplastics may present long-term risks to biodiversity and endemic plant and animal species in such areas. In this study, we used a before-after-control-impact approach to quantify and characterise MP emissions from clothing and shoe outsoles during trail running events. Microplastic deposition on trail surfaces was assessed using both a controlled study and during two public trail running events in New South Wales, Australia (the Duval Dam Buster and the Washpool World Heritage Trail Race). Microplastics were present on trails after all events and included fibres and rubber fragments. Microplastic counts varied considerably depending on trail surface hardness and gradient, and clothing and footwear properties. The controlled study showed running tights (leggings) and shoes with soft rubber outsoles produced more MPs than shirts and hard rubbers. In the trail running events, abrasive wear to shoe outsoles produced an average of 0.3 ± 0.1 to 0.9 ± 0.2 MPs/linear metre/runner, and clothing produced 0.7 ± 0.3 to 2.0 ± 0.3 fibres/linear metre/runner, with fibres accounting for 63–69% of MPs. Microplastic deposition from both footwear and clothing was higher on sloped and rock trail surfaces than flat and soil surfaces. Laser Direct Infrared (LDIR) Imaging indicated the main types of MPs present on trails were polyurethane, polyethylene terephthalate and polyamide. Trail running is increasing in popularity and large-scale events may cause a rapid and significant input of MPs in protected areas. Land managers, event coordinators and outdoor apparel manufacturers could mitigate MP impacts however, by diverting foot traffic around ecologically sensitive areas, capping participant numbers, and developing abrasion resistant clothing and footwear.