Abstract Background Patients with Angina and unobstructed coronary arteries (ANOCA) represent a diagnostic challenge. Current ESC guidelines on the management of patients with chronic coronary syndrome recommend intracoronary spasm testing and coronary spasm represents a frequent diagnosis in such patients. Previous studies have shown that coronary artery flow assessments during acetylcholine testing frequently show non-uniform results. Purpose The aim of the present study was to assess changes in TIMI Frame Count (a quantitative method for the assessment of coronary artery flow) pre- and post- intracoronary acetylcholine testing in patients with ANOCA. Methods From 2007 to 2018, a total of 141 consecutive patients (51.8% female, mean age 61 ± 13.2 years) were included in the study. Among them, 63 patients (45%) presented with myocardial infarction with unobstructed coronary arteries, while 78 patients (55%) presented with stable angina and unobstructed coronaries. Following diagnostic angiography, all patients underwent intracoronary acetylcholine testing using a standardized protocol. TIMI frame count (TFC) was measured both for LAD and LCX before and after ACh injection, and post nitroglycerine injection. All counted frames were adjusted to a frame rate of 30 frames per second (fps). Furthermore, TFC of the LAD was divided by 1.7 to obtain the corrected TFC (cTFC). Results Of all 141 patients, coronary spasm was found in 98 patients (70%) during ACh testing (44% epicardial and 56% microvascular spasm). In the spasm cohort, the median cTFC in the LAD pre-ACh was 21.8 fps (quartiles: 16.3 – 30.0), decreased to 12.9 fps (8.8 - 19.2) post-ACh, and increased to 15.9 fps (10.6 - 21.3) post-nitro. The difference in the LAD between pre-ACh and post-ACh was statistically significant (p < 0.001). In the LCX the median TFC was 26.5 fps (15.0 – 40.5) pre-ACh, decreased to 16.0 fps (11.5 - 28.5) post-ACh and reached 24.0 fps (15.5 - 33.3) post-nitro. Again, this reduction from pre-ACh to post-ACh was statistically significant (p < 0.001). In the negative spasm cohort median cTFC for the LAD was 22.9 fps (16.0 – 18.2) pre-Ach, 15.3 fps (9.4 – 24.7) post-ACh and 15.8 fps (10.6 - 24.7) post-nitro, TFC for LCX was 24.0 fps (18.0 - 36.0) pre-ACh, 18.0 fps (12.0 – 33.0) post-ACh and 24.0 fps (12.0 – 34.0) post-nitro. The change in cTFC/TFC pre-ACh compared to post-ACh was statistically significant for both coronary arteries (LAD, p=0.012; LCX p=0.005, Table 1). Interestingly, in the spasm cohort 22% of LAD and 32% of LCX vessels showed higher TFC post-ACh compared to pre-ACh (Figure 1). Conclusions In patients with ANOCA and coronary spasm assessment of TIMI frame count reveals a hetereogeneous response. The majority of cases shows a decrease in TFC suggesting an increase in flow despite coronary spasm. This interesting finding allows speculations about the presence of coronary shunts.Figure 1