Objectives We performed this study to evaluate the variability of fractional flow reserve (FFR) values which were measured from various methods of hyperemia induction. Background Concerns have been raised regarding the variability of FFR due to different routes for hyperemic agent administration and different hyperemic agents targeting different receptors to induce maximal hyperemia. Methods A total of 656 intermediate coronary lesions from 628 patients with coronary artery disease were analyzed. Among them, 238 lesions underwent FFR measurement with hyperemia induced by both intravenous (IV) and intracoronary (IC) adenosine administration, 318 by IV adenosine/adenosine triphosphate (ATP) and IC nicorandil injection, and 100 by IV adenosine and regadenoson infusion. Results Excellent correlation and close classification agreement (FFR ≤ 0.80) were observed between IV vs. IC adenosine (r = 0.980, CA = 92.9%, Cohen's Kappa = 0.887, P < 0.001), between IV adenosine/ATP vs. IC nicorandil (r = 0.962, CA = 91.2%, Cohen's Kappa = 0.817, P < 0.001), and between IV adenosine vs. regadenoson (r = 0.990, CA = 100%, Cohen's Kappa = 1.000, P < 0.001). When changes in blood pressure (ΔBP) or heart rate (ΔHR) were compared with changes in FFR (ΔFFR) between IV adenosine/ATP and IC nicorandil administration, there were no significant correlations between ΔBP and ΔFFR nor between ΔHR and ΔFFR (r = -0.122, P = 0.076; r = 0.036, P = 0.605, respectively). Conclusions This study suggests that the measurement of FFR is reproducible regardless of the hemodynamic changes, hyperemic agents used, or the route of administration.