The long-term goal of being to able to engineer functional skeletal muscle for clinical application is decades away due to limitations such as the lack of vascularisation and innervation. In spite of this, muscle engineered from primary muscle cell cultures are functionally similar to adult skeletal muscle producing positive force frequency and normal length-tension relationships suggesting they could potentially be used as a model to study muscle physiology and function. Being able to engineer muscle from transformed cell lines would enable the study of genetic modifications (transient and/or stable) on muscle function but is currently not possible due to the short lifespan of constructs (<1 wk). The short lifespan is the result of the fibrin being rapidly digested by the transformed cells due to their high levels of plasmin (an enzyme that degrades fibrin). The aim of this study was to optimise conditions for engineering muscle from C2C12 cells. The number of cells, the media, and the composition of the gel were optimised for maximal force production over time. To overcome the high plasmin activity of the cells, we tested 2 methods to decrease the rate of fibrin breakdown: 1) inhibition of plasmin activity through the inhibitor aprotinin; and 2) increasing the number of cross-links in the fibrin gel using the natural cross-linker genipin. Muscles were engineered with synthetic tendons set 12 mm apart to set their length and for the determination of isometric contractile properties by allowing attachment to a custom made force transducer. With aprotinin, the gel had contracted around the anchor points producing a tubular construct after 3-4 days. This occured between 8-10 days in the genipin constructs. Standard differentiation media (DMEM with 2% horse serum (HS)) caused rapid fibrinolysis resulting in failure by 8 days at the highest concentration of aprotinin. When differentiated for only 2 days in 2% HS then moved to media supplemented with 7% FBS, aprotinin constructs could be cultured for 2 wks. Using genipin in place of aprotinin, constructs could be cultured for up to 4 wks. Aprotinin constructs produced peak twitch force (Pt) 12.6 ± 1.8 μN and peak tetanic tension (Po) 20.1 ± 2.1 μN after 7 days with this reducing over the next 5 days (Pt = 4.9 ± 0.5 μN and Po = 6.5 ± 1.4 μN) prior to construct failure. Genipin constructs produced Pt = 11.5 ± 1.9μN and Po = 35.1 ± 10.9 μN after 7 days and maintained force production over time so that at 3 wks constructs generated Pt = 17.8 ± 4.8 μN and Po = 45.6 ± 10.9μN. In conclusion we have engineered skeletal muscles from the C2C12 cell line which form in 10 days, survive in culture for 3 wks and can be used as a tool to study genetic alterations, pharmacological interventions and exercise on muscle physiology and function.