Background: During fractional flow reserve (FFR) measurement, the simple presence of the guiding catheter (GC) within the coronary ostium might create artificial ostial stenosis, affecting the hyperemic flow. We aimed to investigate whether selective GC engagement of the coronary ostium might impede hyperemic flow, and therefore impact FFR measurements and related clinical decision-making. Methods: In the DISENGAGE (Determination of Fractional Flow Reserve in Intermediate Coronary Stenosis With Guiding Catheter Disengagement) registry, FFR was prospectively measured twice (with GC engaged [FFR _eng ] and disengaged [FFR _dis ]) in 202 intermediate stenoses of 173 patients. We assessed (1) whether ΔFFR _eng –FFR _dis was significantly different from the intrinsic variability of repeated FFR measurements (test-retest repeatability); (2) whether the extent of ΔFFR _eng –FFR _dis could be clinically significant and therefore able to impact clinical decision-making; and (3) whether ΔFFR _eng –FFR _dis related to the stenosis location, that is, proximal and middle versus distal coronary segments. Results: Overall, FFR significantly changed after GC disengagement: FFR _eng 0.84±0.08 versus FFR _dis 0.80±0.09, P <0.001. Particularly, in 38 stenoses (19%) with FFR values in the 0.81 to 0.85 range, GC disengagement was associated with a shift from above to below the 0.80 clinical cutoff, resulting into a change of the treatment strategy from medical therapy to percutaneous coronary intervention. The impact of GC disengagement was significantly more pronounced with stenoses located in proximal and middle as compared with distal coronary segments (ΔFFR _eng –FFR _dis , proximal and middle 0.04±0.03 versus distal segments 0.03±0.03; P =0.042). Conclusions: GC disengagement results in a shift of FFR values from above to below the clinical cutoff FFR value of 0.80 in 1 out of 5 measurements. This occurs mostly when the stenosis is located in proximal and middle coronary segments and the FFR value is close to the cutoff value.