Aims: This study aimed to assess the influence of a second guide wire on the diagnostic accuracy of functional parameters of coronary lesion severity. Methods and results: 65 patients with intermediate coronary lesions underwent myocardial perfusion scintigraphy (MPS). Fractional flow reserve (FFR), coronary flow velocity reserve (CFVR) and hyperemic stenosis resistance 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 2 sensor-equipped guide wires. Overall, functional parameters worsened from single-wire to dual-wire. In patients without ischemia by MPS, the good diagnostic performance of FFR, CFVR and HSR decreased significantly (p<0.001) when assessed with dual-wires, increasing the number of false positives. This trend was more pronounced for HSR, since the presence of a second wire both reduced maximal velocity and increased the pressure gradient. Conclusion: The presence of two guide wires especially across a non-significant lesion by MPS 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 guide wire for the evaluation of stenosis severity using combined pressure and velocity measurements.