We have previously shown that metastatic tumour cells behave differently in functional cell-based assays across our proprietary METAssay™ platform, replicating metastasis biology in vitro. Gene expression analysis explains such behavioural changes and highlights significant changes in the cellular cytoskeleton, which prompted us to pursue the mechanobiology of tumour cell lines and patient tumour samples. Cell deformation during a single force-distance measurement by atomic force microscopy (AFM) was performed to understand the stiffness of growing and moving tumour cells across colorectal cancer cell lines and patient samples. Surprisingly, we saw that moving metastatic cells showed increased stiffness, as measured by Young’s modulus, compared to the non-metastatic or less metastatic cells. A similar translation was observed in patient-derived cells, where node-positive cells had a higher Young’s modulus than the node-negative cohort. A better representation of the difference between such metastatic and non-metastatic cells was made by the retract to approach ratio as measured by the distance of the tip movement, which correlated strongly for both cell lines and patient samples. Moving on to shear stress microfluidics assay mimicking a vasculature, we showed that the metastatic cells had lower velocity than the non-metastatic cells across endothelial and lymph node cells. Further analysis revealed that such a decrease in velocity was due to the increased ability of metastatic cells to adhere to the endothelial or lymph node cells, again correlating with the AFM data. Interestingly, a three-dimensional spheroid spread assay, observed by live imaging, showed that the moving cell lines or patient samples, identified by the AFM and microfluidics experiments, had a different migration pattern than the non-metastatic cell types. We summarize that such differential behaviour of moving and non-moving tumour cells from patient samples can be explored as a novel diagnostic method to predict the metastatic probability of primary colorectal tumours.