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For diagnostic purposes, cryofixation of tissues is a daily routine technique to investigate rapidly about the presence of tumours during a surgical procedure in patients. We performed morphometric analysis of cryofixed muscular tissues according to different techniques. About 1,000 muscle fibers and 1,493 nuclei, were automatically examined. After freezing, ice tissue interfaces shrinkage of the cells were present. Liquid isopentane or liquid nitrogen produced a statistical increase of fractal dimension, D, of the ice-tissue interfaces, P < 0.001 respect to the formalin-fixed samples, cryofixation performed inside the cryostat chamber at t = -20°C produced a D value close to the formalin-fixed samples. Shrinkage of the muscle fibers was higher in the samples cryofixed inside the cryostat chamber (P < 0.001). Cryofixation inside cryostat or by liquid nitrogen caused decreases of the nuclei dimensions and altered nuclear morphology (P < 0.01), liquid isopentane appeared not affecting the nuclei of the fibers. Cryofixation inside the cryostat chamber produced the highest shrinkage but it was reduced performing cryofixation in liquid nitrogen or isopentane. Freezing damage inside the muscle cells was absent in the samples cryofixed inside the cryostat, it was present after cryofixation by liquid nitrogen or isopentane. Subcellular components like the nuclei were preserved by isopentane. This paper present, for the first time, an objective method able to quantify and characterize the damages produced by cryofixation in biological sample for intraoperative consultation., (© 2016 Wiley Periodicals, Inc.)