Skeletal muscle mass contributes to strength and power performance. Understanding the cellular and molecular mechanisms that underpin hypertrophy and the types of contraction modalities that induce such a response, will allow for more specific training programmes for the athletic and clinical populations. In vivo models have provided an insight into the basic mechanisms underpinning the hypertrophic and atrophic processes. In vitro models will allow for the investigation of the intrinsic response of the muscle cell to increased or decreased loading, controlling for the role of systemic influences. An established 3D in vitro model was used, to investigate the effect of two mechanical overload regimes. 4m/ml C2C12 mouse myoblasts were seeded in 3ml of type-1 rat tail collagen and plated into standard dimension chamber slides (n= 4 Control (CT), n= 5 Static Load (SL) and n= 3 Ramp Load (RL)). Each chamber held a custom made A-frame and floatation bar at either end to provide attachment points for the gel, to allow longitudinal tension for the alignment of the myoblasts. The constructs were cultured in growth medium (GM, 20% FBS) for 4 days, before inducing differentiation (DM, 2% FBS and 10ng/ml IGF-I). Following a further 10 day maturation period in DM, the constructs were prepared for experimentation. The constructs were transferred to the tensioning culture monitor (t-CM) for the following regimes of acute mechanical overload; SL = 10% strain for 60 mins, RL = continuous increasing load to achieve 10% strain at 60 mins. CT constructs were tethered to the t-CM without stretch. Constructs were sampled immediately at 60 mins for RNA extraction. Transcript changes in MMP9, IGF-I and genes associated with muscle tissue breakdown or reduced protein synthesis; MuRF-1, MAF-Bx and Myostatin were all investigated using qRT-PCR. Significant (ANOVA) increases in MMP9 were found in both SL (16.4-fold) and RL (22.7-fold) compared to CT (p<0.05). IGF-I significantly increased in the SL condition (71.4-fold, p= 0.001) but not in RL (17.4 fold, p>0.05) compared to CT. A mean reduction in Myostatin expression was observed in both SL and RL conditions compared to CT (p>0.05). However, no differences were found in MuRF-1 and MAF-Bx in either experimental condition, suggesting components of the Ubiquitin Proteasome Pathway have not been manipulated in this system. These findings provide corroborating evidence for the early role of MMP9 and IGF-I in response to mechanical overload. A reduction in Myostatin has been shown to contribute to an increase in net protein accretion. Together, these data have suggested that the mechanical overload has induced a molecular response which has been shown to induce a hypertrophic response. This study has provided evidence for the use of an animal-free bio-mimetic in vitro model to investigate the response of skeletal muscle to mechanical overload.