Background: Vasoreactivity testing with high-dose acetylcholine is considered vasospasm provocation and low-dose as endothelial function testing. Aims: To assess the changes in reaction to low- and high-dose acetylcholine in the endotypes of CAS as defined by the Coronary Vasomotor Disorders International Study Group (COVADIS) working group. Methods: Changes in coronary epicardial diameter, coronary blood flow (CBF) and vascular resistance were determined at low-dose acetylcholine. Results: A total of 88 ANOCA patients were included in this analysis. In the negative group (n = 14) incremental infusion of acetylcholine produced a progressive increase in CBF (p = 0.008). In reaction to low-dose acetylcholine, the epicardial vasospasm group (n = 30) is characterised by epicardial vasoconstriction that is significantly more severe compared to the microvascular vasospasm group (p = 0.004)(n = 23). The equivocal group (n = 21) is characterised by an increase in CBF and reduction in vascular resistance that are both significantly different compared to the epicardial vasospasm group (p = 0.036 and p = 0.007, respectively). High-dose acetylcholine decreased epicardial diameter and CBF significantly in the epicardial vasospasm, microvascular vasospasm and in the equivocal group (all p