The relationship between the Functional Aortic Annulus (FAA), consisting of the Ventriculo-aortic junction (VAJ) and the Sinotubular Junction (STJ) and aortic valve function is incompletely understood. We examined this relationship in a clinically relevant, high fidelity porcine model using a left heart simulator with 3-D echocardiography, hemodynamic assessment, and finite element modeling and by modifying aortic root geometry. Baseline hemodynamic measurements were performed on fresh porcine aortic roots (n=6) on a left heart simulator. VAJ dilatation was performed by remodeling the VAJ and sewing in an oversized Darcon tube graft (diameter=30mm). After dilated VAJ roots were analyzed, the sinotubular junction (STJ) was enlarged by making three veritical incisions at the STJ and patching these areas with three diamond (1cmX1cm) shaped pericardial patches. This created three dependent experimental groups: A) control, B) VAJ dilatation, and C) VAJ + STJ dilatation. Hemodynamic parameters included regurgitant fraction, regurgitant volume, and left ventricular work. 3-D echocardiography measured coaptation surface area. Finite element modeling using (n=10) patient aortic root geometry was performed to determine cusp stresses after each intervention. Baseline VAJ and STJ diameters were 24.3mm interquartile ranges [IQR= 23.3, 24.3] and 24.3mm [IQR=23.3, 25.1]. Following surgical remodeling the VAJ increased to 30mm [IQR=30, 30] and the STJ to 29.8mm [IQR=27.8, 29.8] (p = 0.02). All hemodynamic assessments were similar across groups with the exception of left ventricular work which significantly increased from baseline to VAJ + STJ dilatation (+12%; p=0.03). Regurgitation fraction increased from 4.3% [IQR=3.2, 5.5] at baseline to 14.5% [IQR=13.4, 17.3] (p=0.03) with VAJ dilatation and increased further with STJ dilatation to 18.5% [IQR=10.3, 27.8] (p=0.02). Coaptation surface area decreased from 1.75cm2 [IQR=1.65, 2.00] to 1.23cm2 [IQR=1.05, 1.37] (p=0.02) with VAJ dilatation and further with STJ dilatation to 1.09cm2 [IQR= 1.03, 1.14] (p=0.02 for control and VAJ intervention). An inverse linear relationship was found between VAJ diameter and coaptation surface area (R2=0.70; p=0.001; Figure 1). The combined leaflet stresses in the unaltered root was 530kPa[IQR=380,630] after 4mm diameter VAJ dilatation the stresses increased to 650 kPa [IQR=540,820] (p= 0.001). With an additional 4mm diameter STJ dilatation the leaflet stresses increased to 790 kPa [IQR=600, 1000] (p=0.005). VAJ dilatation produces a graded decrease in leaflet coaptation surface area along with increased leaflet stresses which is further amplified with STJ dilatation. This study provides new insights into mechanisms of annulus mediated AI and their responses to leaflet function and stresses.