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Cardiomyocyte hypertrophy, characterized by an increase in cell size, is associated with various cardiovascular diseases driven by factors including hypertension, myocardial infarction, and valve dysfunction. In vitro primary cardiomyocyte culture models have yielded numerous insights into the intrinsic and extrinsic mechanisms driving hypertrophic growth. However, due to limitations in current approaches, the dynamics of cardiomyocyte hypertrophic responses remain poorly characterized. In this study, we evaluate the application of the Holomonitor M4 digital holographic imaging microscope to track dynamic changes in cardiomyocyte surface area and volume in response to norepinephrine treatment, a model hypertrophic stimulus. The Holomonitor M4 permits non-invasive, label-free imaging of three-dimensional changes in cell morphology with minimal phototoxicity, thus enabling long-term imaging studies. Untreated and norepinephrine-stimulated primary neonatal rat cardiomyocytes were live-imaged on the Holomonitor M4, which was followed by image segmentation and single-cell tracking using the HOLOMONITOR App Suite software version 4.0.1.546. The 24 h treatment of cultured cardiomyocytes with norepinephrine increased cardiomyocyte spreading and optical volume as expected, validating the reliability of the approach. Single-cell tracking of both cardiomyocyte surface area and three-dimensional optical volume revealed dynamic increases in these parameters throughout the 24 h imaging period, demonstrating the potential of this technology to explore cardiomyocyte hypertrophic responses with greater temporal resolution; however, technological limitations were also observed and should be considered in the experimental design and interpretation of results. Overall, leveraging the unique advantages of the Holomonitor M4 digital holographic imaging system has the potential to empower future work towards understanding the molecular and cellular mechanisms underlying cardiomyocyte hypertrophy with enhanced temporal clarity., Competing Interests: Conflicts of Interest: The authors declare no conflicts of interest.