Solid tumours are complex and heterogeneous systems, which exist in a dynamic biophysical microenvironment. Conventional cancer research methods have long relied on two-dimensional (2D) static cultures which neglect the dynamic, three-dimensional (3D) nature of the biophysical tumour microenvironment (TME), especially the role and impact of interstitial fluid flow (IFF). To address this, we undertook a transcriptome-wide analysis of the impact of IFF-like perfusion flow using a spheroid-on-chip microfluidic platform, which allows 3D cancer spheroids to be integrated into extracellular matrices (ECM)-like hydrogels and exposed to continuous perfusion, to mimic IFF in the TME. Importantly, we have performed these studies both in experimental (normoxia) and pathophysiological (hypoxia) oxygen conditions. Our data indicated that gene expression was altered by flow when compared to static conditions, and for the first time showed that these gene expression patterns differed in different oxygen tensions, reflecting a differential role of spheroid perfusion in IFF-like flow in tumour-relevant hypoxic conditions in the biophysical TME. We were also able to identify factors primarily linked with IFF-like conditions which are linked with prognostic value in cancer patients and therefore could correspond to a potential novel biomarker of IFF in cancer. This study therefore highlights the need to consider relevant oxygen conditions when studying the impact of flow in cancer biology, as well as demonstrating the potential of microfluidic models of flow to identify IFF-relevant tumour biomarkers.