["The Journal of Physiology, Volume 604, Issue 7, Page 2867-2897, 1 April 2026. ", "\nAbstract figure legend Gait analysis reveals neuromuscular dysfunction following hindlimb suspension and partial recovery after reloading. Schematic overview of the experimental design and principal findings. Mice underwent 14 days of hindlimb suspension (HLS), resulting in muscle atrophy marked by reduced fiber size, satellite cell loss, and decreased gastrocnemius contractility. After 7 days of reloading, structural and functional recovery remained incomplete. In vivo gastrocnemius contractility was markedly decreased after HLS and only partially restored with reloading. Treadmill‐based gait analysis revealed domain‐specific impairments across neural rhythm control, neuromuscular coordination, and postural stability, while strength‐related parameters such as paw drag exhibited mild but consistent alterations. Molecular analysis identified sustained MG29 upregulation, MG53 redistribution, and dysregulated expression of neuromuscular genes (e.g., Ninj1, Prkg1, Ryr1, S100a1), indicating disrupted muscle plasticity and synaptic remodeling. These findings support gait analysis as a valuable tool for assessing therapeutic strategies aimed at preserving or restoring mobility. Created in BioRender. Lee, S. (2025) https://BioRender.com/4jsuvwv.\n\n\n\n\n\n\n\n\n\nAbstract\nDisuse‐induced skeletal muscle atrophy, commonly resulting from bedrest, immobilisation or spaceflight, leads to rapid loss of muscle mass and impaired mobility. Although muscle mass and contractile force are standard assessments in experimental models, these measures often fail to capture neuromuscular co‐ordination deficits essential for effective movement. To better characterise these deficits, we employed a mouse hindlimb suspension (HLS) model for 14 days to induce disuse atrophy, confirmed by reductions in muscle mass, fibre type remodelling and satellite cell depletion, all of which were only partially reversed after a 7‐day reloading period. In vivo analysis showed that gastrocnemius contractile force was significantly reduced following HLS and recovered incompletely after reloading. To functionally assess mobility, we implemented a non‐invasive treadmill‐based gait analysis, which revealed domain‐specific impairments across neural control/rhythm, neuromuscular co‐ordination and stability/variability, which were only partially restored after reloading, whereas muscle strength‐related metrics such as paw drag showed mild but consistent alterations. At the molecular level, we identified elevated expression of MG29, subcellular redistribution of MG53 and altered expression of neuromuscular function‐related genes (e.g. Ninj1, Prkg1, Ryr1 and S100a1), suggesting that MG29 and MG53 may contribute to impaired muscle plasticity and synaptic remodelling. Overall, our findings demonstrate that gait analysis can enhance the functional assessment of muscle disuse and recovery, offering a translational tool to evaluate interventions targeting atrophy‐related mobility decline.\n\n\n\n\n\n\n\n\n\nKey points\n\nHindlimb suspension induces muscle atrophy and contractile loss, but functional consequences are not fully captured by traditional measurements.\nGait analysis provides a non‐invasive framework to evaluate neuromuscular performance across four domains: muscle strength/size, neural control/rhythm, neuromuscular co‐ordination and stability/variability. Hindlimb suspension caused domain‐specific impairments in rhythm control, co‐ordination and stability, which were only partially restored after reloading, whereas strength‐related metrics such as paw drag showed mild but consistent alterations.\nCorrelation analyses revealed parallel reductions in propulsion‐ and rhythm‐related gait metrics alongside decreases in muscle fibre size and tetanic force, indicating a functional–structural linkage between gait output and muscle integrity.\nFunctional impairment is associated with satellite cell loss, MG29 upregulation, MG53 redistribution and neuromuscular function‐related gene alteration.\nThese findings identify gait metrics as biomarkers that may serve as early, non‐invasive indicators of muscle disuse and recovery, providing mechanistic insights and a new tool to evaluate interventions targeting atrophy‐related mobility loss.\n\n\n"]