The objective of this study was to determine the effects of temporal hydrostatic pressure (HP) on the properties of scaffoldless bovine articular cartilage constructs. The study was organized in three phases:​ First, a suitable control for HP application was identified. Second, 10 MPa static HP was applied at three different timepoints (6–10 days, 10–14 days, and 14–18 days) to identify a window in construct development when HP application would be most beneficial. Third, the temporal effects of 10–14-day static HP application, as determined in phase II, were assessed at 2, 4, and 8 weeks. Compressive and tensile mechanical properties, GAG and collagen content, histology for GAG and collagen, and immunohistochemistry for collagen types I and II were assessed. When a culture control identified in phase I was used in phase II, HP application from 10 to 14 days resulted in a significant 1.4-fold increase in aggregate modulus, accompanied by an increase in GAG content, while HP application at all timepoints enhanced tensile properties and collagen content. In phase III, HP had an immediate effect on GAG content, collagen content, and compressive stiffness, while there was a delayed increase in tensile stiffness. The enhanced tensile stiffness was still present at 8 weeks. For the first time, this study examined the immediate and long-term effects of HP on biomechanical properties, and demonstrated that HP has an optimal application time in construct development. These findings are exciting as HP stimulation allowed for the formation of robust tissue-engineered cartilage;​ for example, 10 MPa static HP resulted in an aggregate modulus of 273 ± 123 kPa, a Young's modulus of 1.6 ± 0.4 MPa, a GAG/wet weight of 6.1 ± 1.4%, and a collagen/wet weight of 10.6 ± 2.4% at 4 weeks.