Purpose: Isotemporal substitution analysis offers new insights for public health, but has only recently been applied to sedentary behavior research. We aimed to quantify associations between the substitution of 10 minutes of sedentary behavior with 10 minutes of light physical activity (LPA) or moderate-to-vigorous physical activity (MVPA) and the prevalence of cardiovascular disease (CVD). Age was also explored as a potential effect modifier. Methods: We completed a secondary analysis of data from 1477 adults from the Health Survey for England (2008). Sedentary time, LPA and MVPA were measured using accelerometry. We applied isotemporal models to quantify the relationship with CVD prevalence of replacing 10 minutes of sedentary time with equivalent amounts of LPA or MVPA. Prevalence risk ratios (RR) with 95% confidence intervals (CI) are presented, adjusted for covariates. The role of age as an effect modifier was explored via age × MVPA and age × LPA interactions. CVD was defined as per the International Classification of Diseases. Results: The prevalence of CVD was 24%. The RR was 0.97 (95% CI: 0.96 to 0.99) for LPA and 0.88 (0.81 to 0.96) for MVPA. Substitution of approximately 50 minutes of LPA would be required for an association equivalent to 10 minutes of MVPA. The beneficial association of MVPA was attenuated with age, with a decrease in the relative risk reduction of ~7% per decade. Conclusions: Isotemporal substitution of sedentary time with LPA was associated with a trivial relative risk reduction for CVD, whereas the equivalent replacement with MVPA had a small beneficial relationship. With respect to CVD prevalence, MVPA might become decreasingly important in older individuals. Prospective studies are needed to investigate causality.