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Introduction: There is little experimental evidence of the impact of multi-use recreational trails that support physical activity on cardiovascular disease (CVD). Hypothesis: Neighbourhoods that added multi-use trails would experience a greater decline in CVD events and risk factors compared to neighbourhoods that did not. Methods: We used a difference in differences design to study the addition of four multi-use trails 4-7km in length on CVD-related outcomes using administrative health, census and built environment data available for all citizens 30 years of age and older from Winnipeg, Canada. A 400m buffer stratified intervention and comparison neighbourhoods. Bicycle counts were recorded via electromagnetic counters for 5 years on all trails. The primary and secondary outcomes were composite measures of incident CVD events (mortality, ischemic heart disease, cerebrovascular disease and congestive heart failure) and CVD risk factors (hypertension, diabetes and dyslipidemia) and assessed quarterly for 10 years prior to and 6 years following the intervention. Intervention and comparison areas were propensity score matched, using scores regressed from baseline measures of age, sex, socioeconomic indicators, and built environment attributes that support physical activity. Results: Between 2012 and 2018, 1,429,588 cyclists were recorded on the trails and cycling use varied ~2.0-fold across the trails. Between 2000 and 2018, there were 82,632 CVD events and 201,058 CVD risk events. During the 18-year natural experiment CVD event rates and risk factors declined ~33% in both comparison and intervention neighbourhoods. In propensity score matched regression models, the incident rate ratio was 1.06 (95% CI: 0.90 to 1.24) for CVD events and 0.92 (95% CI: 0.84 to 1.02) for CVD risk factors. Sensitivity analyses revealed greater effect sizes with increasing trail use (incident rate ratios for highest vs lowest cycling counts = 0.85; 95% CI: 0.75 to 0.96 vs 1.08; 95% CI: 0.92 to 1.27). Conclusions: The addition of recreational multi-use trails was not associated with changes in overall CVD events or risk factors in adjacent neighbourhoods, compared to distant neighbourhoods, however, the effects on CVD risk factors may be influenced by trail use.