In cartilage tissue engineering studies, the stimulatory effect of a constant magnitude of mechanical perturbation declines after the first two weeks of culture. Similarly, it is known that chondrocyte-agarose constructs should not be loaded within the first days after seeding, to prevent considerable cell death, suggesting a mechanical threshold. This study aims to establish a relationship between chondrocyte deformation and death, and to evaluate the protective effect of the pericellular matrix (PCM) that is formed in 3D cultures. Chondrocyte viability was monitored every hour for 24 hours after applying a strain range of 0% to 25% to agarose constructs containing chondrocytes, cultured for 1, 3, 5, 7 or 10 days. At these culture time points, PCM thickness and chondrocyte deformation were assessed by means of histology and assayed for biochemical contents. Inverse finite element simulations were used to evaluate the change of mechanical properties of the chondrocyte and PCM over the 10 day culture duration. Chondrocyte death was demonstrated to be dependent on both the magnitude and duration of straining. The highest cell death was observed at day 1 (43%), reducing over culture duration (15% at day 3, and 2.5% at day 10). Cell deformation at 25% compression decreased significantly over culture duration (aspect ratio of 2.24 ± 0.67 at day 1 and 1.45 ± 0.24 at day 3) and with increased matrix production. Inverse finite element simulations showed an increasing PCM Young’s modulus of 45 KPa at day 3 to 162 KPa at day 10. The current results provide evidence for a mechanical threshold for chondrocyte death and for the protective effect of the PCM. As such, these insights may help in establishing mechanical loading protocols for cartilage tissue engineering studies.