Background: Current tissue-engineered cartilage constructs contain insufficient amounts of collagen, whose function is to resist tension. We postulate that dynamic tension is necessary to stimulate collagen formation. Another shortcoming is that TE cartilage does not possess native zonal variations. We hypothesize that applying depth-varying mechanical cues would stimulate ECM synthesis depth-dependently. We developed a dedicated loading regime called ‘sliding indentation’ which enables us to apply dynamic tension as well as depth-varying strain fields to chondrocyte-seeded agarose constructs. Objective: In 2 study designs, we explored whether sliding indentation would increase collagen content and induce depth-varying ECM distribution. Methods: In the first study, we developed an ‘agarose-sandwich model’ which involves embedding of a thin chondrocyte-seeded 0.5% agarose layer between two cell-free 3% agarose layers. In the second study, 3 mm thick chondrocyte-seeded agarose constructs were created. Sliding indentation at 10% depth and 1 Hz was applied to constructs in both studies for 4h/day during 28 days and unloaded constructs served as control. Results: Sliding indentation resulted in an increased amount of collagen in the produced cartilage layer. Furthermore, sliding indentation for 7 days resulted in a depth-dependent response at gene expression levels, with highest response in regions that received highest strains. Analysis of protein expression after 28 days showed a similar depth-dependent distribution in all constructs, which further enhanced by sliding indentation. Conclusions: Sliding indentation can increase collagen content and enhances depth-dependent ECM distribution and is therefore a promising strategy for culturing cartilage with improved properties.