This study investigated the effects of multiple freeze-thaw (F-T) cycles on water mobility, microstructure damage and protein structure changes in porcine longissimus muscle. The transverse relaxation time T2 increased significantly when muscles were subjected to multiple F-T cycles (P<0.05), which means that immobile water shifted to free water and the free water mobility increased. Multiple F-T cycles caused sarcomere shortening, Z line fractures, and I band weakening and also led to microstructural destruction of muscle tissue. The decreased free amino group content and increased dityrosine in myofibrillar protein (MP) revealed that multiple F-T cycles caused protein cross-linking and oxidation. In addition, the results of size exclusion chromatography, circular dichroism spectra, UV absorption spectra, and intrinsic fluorescence spectroscopy indirectly proved that multiple F-T cycles could cause protein aggregation and degradation, α-helix structure disruption, hydrophobic domain exposure, and conformational changes of MP. Overall, repeated F-T cycles changed the protein structure and water distribution within meat. •An increased T2 means the F-T cycles caused immobile water to shift to free water.•Multiple F-T cycles caused protein cross-linking, degradation and oxidation.•The microstructure of muscle tissue was disrupted by multiple F-T cycles.•Repeated F-T cycles had a detrimental effect on the protein structure and WHC.