Measurements of apparent diffusion coefficient (ADC) using magnetic resonance imaging (MRI) have been suggested as potential imaging biomarkers for monitoring tumor response to treatment. However, conventional pulsed-gradient spin echo (PGSE) methods incorporate relatively long diffusion times, and are usually sensitive to changes in cell density and necrosis. Diffusion temporal spectroscopy using the oscillating gradient spin echo (OGSE) sequence is capable of probing short length scales, and may detect significant intracellular microstructural changes independent of gross cell density changes following anti-cancer treatment. To test this hypothesis, SW620 xenografts were treated by barasertib (AZD1152), a selective inhibitor of Aurora B kinase which causes SW620 cancer cells to develop polyploidy and increase in size following treatment, ultimately leading to cell death through apoptosis. Following treatment, the ADC values obtained by both the PGSE and low frequency OGSE methods increased. However, the ADC values at high gradient frequency (i.e. short diffusion times) were significantly lower in treated tumors, consistent with increased intracellular restrictions/hindrances. This suggests that ADC values at long diffusion times are dominated by tumor microstructure at long length scales, and may not convey unambiguous information of subcellular space. While the diffusion temporal spectroscopy provides more comprehensive means to probe tumor microstructure at various length scales. This work is the first study to probe intracellular microstructural variations due to polyploidy following treatment using diffusion MRI in vivo. It is also the first observation of post-treatment ADC changes occurring in opposite directions at short and long diffusion times. The current study suggests that temporal diffusion spectroscopy potentially provides pharmacodynamic biomarkers of tumor early response which distinguish microstructural variations following treatment at both the subcellular and supracellular length scales.