Given that diffusion tensor imaging (DTI) may be able to provide additional information beyond T1 and T2 relaxation times regarding tissue anisotropy and microstructure, the aim of this study was to investigate DTI, T1 and T2 in detecting changes in the nucleus pulposus of the intervertebral disc (IVD) during enzyme digestion and highlight the effect of hydration. Fifty-nine bovine caudal discs were separated into in situ, hydrated and trypsin-digested groups, and subjected to a multi-parametric magnetic resonance imaging (MRI) acquisition followed by confined compression test and biochemical assays. The effects of trypsin digestion versus hydration and of treatment versus duration on the MRI parameters were quantified by ANOVAs. T1 and T2 decreased progressively between the in situ and hydrated groups, increased progressively between the hydrated and digested groups, and did not change between the in situ and digested groups. Fractional anisotropy (FA) increased in the digested groups. The treatment had a significant effect on T1, T2 and FA, while its duration had a significant effect only on FA. FA was the most sensitive magnetic resonance parameter to the trypsin digestion of isolated IVDs. The effect of cleaving of proteoglycans due to trypsin digestion on T1 and T2 was hidden by the effect of hydration. For clinical applications and ex vivo studies, multi-parametric MRI should include DTI sequences to detect tissue-level changes when hydration is present.