Regarding the determination of the biomechanical parameters in a reliable in vitro cell model for diffuse axonal injury (DAI), our study aimed to demonstrate connections between those parameters and secondary axotomy through examination of morphological alterations under a variety of traumatic conditions. An in vitro cell model for DAI was established in primary cultured mouse neurons by uniaxial mechanical stretching of non-myelinated axons under various traumatic conditions: strain (epsilon) = 5, 10, 20, and 50%; strain time (t) = 500, 100, and 20 ms; strain rate ranging between 0.1 and 25 s(-1). Axonal real strains (strain(axon)) were measured as 4.53 +/- 0.27, 9.02 +/- 0.91, 17.75 +/- 1.65, and 41.8 +/- 4.4%. Axonal real strain rates (SRaxon) ranged between 0.096 +/- 0.0054 and 20.9 +/- 2.2 s(-1). Results showed there was no obvious abnormality of axons with a lower strain condition (strain(axon) < 17.75 +/- 1.65%) during the acute phase within 30 min after injury. In contrast, acute axonal degeneration (AAD) was observed in the axons following injury with a higher strain condition (SRaxon > 17.75 +/- 1.65%). In addition, the incidence and degree of AAD were closely correlated with strain rate. Specifically, AAD occurred to all axons that were examined, when + = 50% (strainaxon = 41.8 +/- 4.4%) for 20 ms, while no spontaneous rupture was observed in those axons. Besides, the concentration of Ca2+ within the axonal process was significantly increased under such traumatic conditions. Moreover, the continuity of axon cytoskeleton was interrupted, eventually resulting in neuronal death during subacute stage following injury. In this study, we found that there is a minimum strain threshold for the occurrence of AAD in non-myelinated axons of primary cultured mouse neurons, which ranges between 9.02 +/- 0.91 and 17.75 +/- 1.65%. Basically, the severity of axonal secondary axotomy post DAI is strain rate dependent under a higher strain above the threshold. Hence, a reliable and reproducible in vitro cell model for DAI was established, when epsilon = 50% (strain(axon) = 41.8 +/- 4.4%) for 20 ms.