Mechanical stimuli have been shown to affect cell behaviour in terms of proliferation, apoptosis, and protein expression. In terms of cardiovascular diseases, for example, endothelial and smooth muscle cells exposed to an abnormal strain environment have been associated with atherosclerosis and in-stent restenosis. The FX-4000 system (Flexercell Tension Plus System, Flexcell Corporation, McKeesport, Pennsylvania, USA) is an in-vitro system that is widely used to strain cells in order to evaluate their response to strain. The precision, accuracy, and repeatability of the strains controlled by the system are therefore crucial to analyse and interpret the results confidently. The aim of this study was to investigate the mechanical behaviour of the FX-4000 Flexercell six-well-plate silicon membranes for static and dynamic cyclic strains by measuring the maximum peak strain and analysing the change in the membrane deformation after cyclic strain for 0 h, 24 h, and 48 h at different strain amplitudes and frequencies. The results of the tests conducted demonstrate notable differences between the measured strains of the membranes in comparison with both the inputs and the outputs of the Flexcell software. The calibration method used by Flexcell International assumes that the strain values determined for a given vacuum pressure on the silicone membranes are reliable for different waveforms and frequencies. The data reported here clearly indicate that this is not the case. The results indicate that a unique calibration pressure-strain curve must be determined for each test given the viscoelastic nature of the Flexcell system. A new method to calibrate the machine in house was applied using new pressure-strain equations. This new calibration method has been presented and should enable researchers using the Flexcell machine to set up their cell experiments more accurately.