Background Impaired autoregulation may contribute to the pathogenesis of cerebral small vessel disease (SVD). Reliable protocols for measuring microvascular reactivity are required to test this hypothesis and for providing secondary endpoints in clinical trials. Aims To develop and assess a protocol for acquisition and processing of cerebrovascular reactivity (CVR) by MRI, in subcortical tissue of patients with SVD and minor stroke. Methods We recruited 15 healthy volunteers, testing paradigms using 1- and 3-minute 6% CO2 challenges with repeat scanning, and 15 patients with history of minor stroke. We developed a protocol to measure CVR and delay times, assessing tolerability and reproducibility in grey and white matter (GM, WM) areas. Results The 3-minute paradigm yielded more reproducible data than the 1-minute paradigm (CV respectively: 7.9–15.4% and 11.7–70.2% for CVR in GM), and was less reproducible in WM (16.1–24.4% and 27.5–141.0%). Tolerability was similar for the two paradigms, but mean CVR and CVR delay were significantly higher for the 3-minute paradigm in most regions. Patient tolerability was high with no evidence of greater failure rate (1/15 patients vs. 2/15 volunteers withdrew at the first visit). GM CVR was lower in patients than in volunteers (0.110—0.234 vs. 0.172—0.313 %/mmHg; p<0.05 in 6/8 regions), as was the WM CVR delay (16.2—43.9 vs. 31.1—47.9 s; p<0.05 in 4/8 regions). Conclusions An effective and well-tolerated protocol for measurement of CVR was developed for use in ongoing and future trials to investigate SVD pathophysiology and to measure treatment effects.