Background. Non-traumatic bicycle injuries are common. However, research available on non-traumatic injuries in amateur cyclists is more than a decade old, and most of the research on this topic has been done in Europe and America on professional cyclists in multi-day cycling events. An understanding of the common injuries may lead to appropriate prevention intervention. Objective. To determine the incidence of overuse injuries in amateur cyclists preparing for participation in a 1-day cycle challenge. Methods. A questionnaire was emailed to participants of the 2012 Momentum 94.7 Cycle Challenge, which amateur participants ≥18 years old were invited to complete. Data on demographics, training habits and the participants’ injury profile in the preceding year were collected. Results. Of the 3 300 respondents, 75% were male and 59% were between 30 and 50 years old. Non-traumatic injury, pain or neurological symptoms were reported by 88% of the respondents. The percentages of all respondents who experienced problems in the following anatomical areas were as follows: neck 34%, back 41%, hand/wrist 41%, buttock/perineum 41%, hip 7%, knee 33% and foot/ankle 24%. Knee pain was responsible for the need to stop training for the largest percentage of respondents. Neurological complaints were common in respondents who experienced neck, back, hand/wrist, buttock/perineum and foot/ankle problems. Conclusion. Non-traumatic injuries in amateur cyclists are common, with back, hand/wrist and buttock/perineal symptoms the most frequent problems. Knee problems caused the greatest need to stop training and seek medical help.