Assessment of replenishment kinetics (RK) following ultrasound-induced destruction of contrast microbubbles allows quantification of myocardial blood flow reserve (MBFR) applying the modelf (t) = A (1 − e-ßt), with parameter β describing mean flow velocity and parameter A representing blood volume. However, few data on the variability and reproducibility of RK in a clinical setting are available. Therefore, we examined 30 patients in a rest—adenosine protocol in one center. Off-line quantification of real-time perfusion imaging (RTPI) and triggered replenishment imaging (TRI) was performed at two sites and compared with coronary angiography and flow reserve measurements. Parameter A was found to be robust in all investigated segments (coefficient of variation (CV) < 7.2%± 5.1). Variability was lowest for parameter β using TRI in apical segments (CV 6.5%± 5.2, P < 0.01). Highest CV was found with RTPI in lateral segments (CVβ: 39.8%± 40.6). Concerning day-to-day reproducibility both methods revealed similar results within range of heterogeneity of myocardial blood flow. Both sites obtained significantly lower MBFR in patients with flow-limiting CAD, compared to subjects without (P < 0.01). Correlation of both sites showed close relationship (y = 0.88x + 0.45, r = 0.83, P < 0.0001), without systematic bias. TRI significantly reduces variability of RK in quantitative MCE. Assessment of MBFR allows investigator-independent evaluation of CAD.