The aim of this study was to assess the influence of a second guidewire on the diagnostic accuracy of functional parameters of coronary lesion severity. Sixty-five patients with intermediate coronary lesions underwent myocardial perfusion scintigraphy. Fractional flow reserve (FFR), coronary flow velocity reserve (CFVR), and hyperemic stenosis resistance (HSR) index (HSR = stenosis pressure gradient / velocity) were determined in 77 lesions. Distal pressure and velocity were acquired simultaneously (dual wire) and sequentially (single wire) with two sensor-equipped guidewires. Overall, functional parameters deteriorated from single- to dual-wire assessment. In patients without ischemia, the good diagnostic performance of FFR, CFVR, and HSR deteriorated significantly (P < 0.001) when assessed by dual wires, with an increase in the number of false-positive results. This trend was more pronounced for HSR, since the presence of a second wire reduced maximal velocity and increased the pressure gradient. The presence of two guidewires, especially across a myocardial perfusion scintigraphy-induced nonsignificant lesion, is associated with overestimation of the hemodynamically assessed lesion severity and, therefore, is likely to have a major impact on clinical decision making. This underscores the advantage of a dual-sensor-equipped guidewire for the evaluation of stenosis severity by combined pressure and velocity measurements.