We studied biomembrane fluctuations by calculating the instantaneous shape of model membranes adhered to micro-patterned substrates, using micro-interferometry. The model consisted of partially adherent giant unilamellar vesicles (GUVs) which were osmotically deflated. Adhesion was effected via the specific ligand–receptor interaction of biotin–neutravidin. Special micro-structured adhesive substrates were developed where the receptors were distributed in the form of grids or lines. Dual-wavelength reflection interference contrast microscopy (DW-RICM) measurements revealed that on the structured adhesive substrates GUVs exhibit regions of bound and fluctuating membrane, in accordance with the underlying pattern. In the fluctuating zone, the membrane presented itself as a flat-topped hill for an initial osmotic difference of 70 mOsm l−1. The membrane–substrate distance saturated at a plateau of 79 ± 9 nm. In this plateau, the fluctuation amplitude was found to be 10 ± 3 nm. Variation of the shape (grid versus lines) or size (grids of 3.5 or 7 μm lattice constant) influenced neither the height nor the fluctuation amplitude in the plateau. Fourier analysis revealed that the mode corresponding to a wavelength of twice the pattern size always contributed, and, depending on the substrate, additional modes were sometimes present. The plateau height could be tuned from 0 to 538 nm by changing the initial osmotic gradient between the inside and outside of the GUV, which effectively tuned the membrane tension. The corresponding fluctuation amplitude ranged from non-detectable to a maximum of 17 nm. Our results can be interpreted in the light of a tension dependent effective interaction potential.