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Fractured crystalline rocks are widely regarded as suitable host formations for waste isolation applications, including deep geological repositories for spent nuclear fuel. Fluid flow in these systems is predominantly controlled by networks of fractures whose hydraulic properties are governed by internal heterogeneity, contact area distribution, and stress-dependent aperture evolution. These properties are strongly influenced by in situ stress conditions, which may evolve over geological to engineering time scales due to processes such as glacial loading, stress redistribution, and excavation-induced damage. Robust representation of fracture-scale flow behaviour is therefore critical for the development and calibration of large-scale discrete fracture network models.In this study, we investigate the coupled hydro-mechanical behaviour of a natural single fracture using a laboratory-scale flow experiment under increasing normal load. The fracture aperture field was reconstructed using high-resolution 3D scanning of the opposing fracture surfaces, with vertical alignment refined using pressure-sensitive film measurements. A systematic sensitivity analysis of 27 alignment cases, incorporating translational uncertainties along the x-, y- and z-directions was performed to quantify their influence on flow behaviour. Measured flow rates were compared against predictions from the local cubic law under varying normal stress. Results demonstrate that flow is highly sensitive to fracture surface alignment, with misalignment along the flow direction and normal direction exerting the dominant influence. The local cubic law systematically overestimates flow by at least two orders of magnitude for all loading cases. Furthermore, the application of a constant correction factor to convert mechanical to hydraulic aperture, calibrated under unloaded conditions, fails to reproduce experimental flow rates as normal load increases. We propose a stress-dependent correction factor linked explicitly to the evolution of fracture contact area. Incorporating this relationship yields close agreement with experimental observations across all loading and alignment cases.