Research surrounding the mechanisms that govern skeletal muscle adaptation to exercise, have begun to be characterised in vivo. Developing an in vitro model of skeletal muscle, which can predictably respond to stimulation in a similar way to in vivo exercise, will combat experimental issues including; subject recruitment and biopsy sampling. This investigation used 3D in vitro skeletal muscle model, to examine the metabolic and transcriptional responses to two diverse stimulations. 4m/ml C2C12 myoblasts were seeded in 3ml of type-1 rat tail collagen and plated into standard dimension chamber slides (n= 3 each for Control (CT), Continuous Cyclic (CC) and Intermittent Cyclic (IC)). Each chamber held an A-frame and floated bar at each end to provide attachment points for the gel, for the alignment of the myoblasts. The constructs were cultured in growth medium (GM, 20% FBS) for 4 days. Following a further 10 day period in differentiation media (DM, 2% FBS and 10ng/ml IGF-I), the constructs were used for experimentation. The constructs were transferred to the tensioning culture monitor (t-CM) for the following regimes of mechanical overload; CC = 7.5% strain continuous cyclic for 60 mins. IC = 10% strain cyclic stretch for 10 reps followed by a 90 sec delay (1 set), a further 3 sets were performed (total = 1 ‘exercise’), with a 3 mins rest between each exercise of which 4 were completed. CT constructs were tethered to the t-CM without stretch. Conditioned media was sampled every 5 mins for the CC condition and post every ‘exercise’ in the IC condition for [Lactate] analysis. Constructs were sampled immediately at 60 minutes for RNA extraction. Transcript changes in metabolically activated associated genes were investigated using qRT-PCR. CC significantly increased [Lactate] from 1.73 ± 0.31 mmol.L to 5.57 ± 0.85 mmol.L after 5 mins (p<0.01). There were no observed significant differences in media [Lactate] between any time-points from 5-60 mins (p>0.05), indicative of a steady state in Lactate production. IC significantly (ANOVA) increased conditioned media [Lactate] from 1.73 ± 0.31 mmol.L to 7.05 ± 2.61 mmol.L after the first ‘exercise’ (p<0.01). No significant increases in [Lactate] were observed for CT across samples. CC reduced the expression of Cytochrome C by 0.23-fold compared to CT, whilst significantly increasing NRF-2 (p<0.05). IC significantly increased PGC-1α, Cytochrome C, NRF-1 and NRF-2 mRNA expression compared to CT (p<0.05). Increases in PGC-1α, Cytochrome C, NRF-1 and NRF-2 mRNA, suggests an adaptation towards increasing the oxidative potential of the tissue. An increase in NRF-2 mRNA despite a moderate, but non-significant change in NRF-1 mRNA in the CC condition proposes a greater role for NRF-2 in the response to CC stimulation. This system can be manipulated to induce responses similar to those seen with in vivo exercise.