Three-dimensional (3D) bioprinting studies the initial condition of printed constructs, which makes construct static, and neglects the dynamic changes in natural tissue conformation during tissue repair, disease progression and regeneration processes. Therefore, the concept of four-dimensional (4D) bioprinting has been introduced that allows for the fabrication of scaffolds that can transform in time. The extracellular matrix stiffness is one component involved in the in vivo dynamic microenvironment of tissues such as cartilage. Nonetheless, the implementation of dynamic stiffness in in vitro 3D cell culture has not been intensively investigated yet, in particular not in 4D bioprinting for cartilage applications. Therefore, the aim of this study is to create a method for the temporal stiffening of a human mesenchymal stem cell-laden scaffold in 4D bioprinting for cartilage applications. To do this, inks made of tyramine-modified hyaluronic acid (THA), and THA combined with sodium alginate (SA) (THA-SA) were studied on their ability to stiffen days after being printed through photocrosslinking or ionic crosslinking (secondary crosslinking). The method that generated the greatest increase in stiffness was evaluated on the effect of time on stiffening and the effect of stiffening on the swelling behaviour and the internal structure of the scaffold. Next, the 4D printing method was translated to 4D bioprinting and the influence of the temporal stiffening method on the mechanical properties, cell viability, gene expression and matrix deposition was determined. This study demonstrated that in 4D printing, secondary photocrosslinking of THA and secondary ionic crosslinking of THA-SA resulted into scaffolds with a significant increase in stiffness. The method for secondary ionic crosslinking of THA-SA (2.5% w/v THA, 1.5% w/v SA) scaffolds achieved the largest increase in stiffness and was able to temporally stiffen at day 1, 3 or 5. This increase in stiffness was associated with a decrease in swelling ratio and a maintained internal microstructure. For the translation to 4D bioprinting, the method was adjusted for cell culture conditions and resulted in an increase in stiffness. However, the stiffening was not yet reproducible and therefore optimalization of the method will be required. Nevertheless, in the first week of cell culture, the bioprinted scaffolds achieved high cell viability and an equal cell distribution. At day 7, the gene for collagen type II was not expressed and cartilage-specific matrix deposition was not detected. With future studies focusing on optimizing the introduced 4D bioprinting protocol, this novel method for the ionic secondary crosslinking of THA-SA may function as a platform for studying the effect of temporal matrix stiffening on the behaviour of the embedded cells in fundamental research or as a stimulus for tissue formation in tissue engineering.