Regulation of skeletal muscle mass and phenotype is critical in order to optimise training and performance in numerous athletic disciplines. However, whilst in vivo models used to examine skeletal muscle regulation and adaptation have given us some understanding of this field, in vitro studies allow for deeper exploration of the processes governing skeletal muscle growth and phenotype. Most in vitro studies in this discipline utilise two dimensional cultures, or use cells from non-human sources, bringing into question the validity of the results when related to humans. The purpose of this work was to temporally examine the maturation and bio-mimetic structure of human muscle derived cells when in both two and three dimensional culture. 4 x 105 human muscle derived cells were seeded into six well plates (2D) or onto a fibrin gel matrix (3D) which self assembled over time (1). The cells were maintained in DMEM with 20% FCS until confluent, at which point constructs were switched to DMEM with 2% FCS and 10ng/ml IGF-1 to encourage myotube formation. At 7, 14 and 21 days RNA was extracted using the TRIzol® method and mRNA expression of myosin heavy chain (MYH) isoforms was detected using RT-qPCR and analysed using the 2-ΔΔCT method. Myotube diameters were measured using image J software and over 30 myotubes were counted from each condition. n=4-6 for each condition. MYH expression was analysed by ANOVA, and morphology by t-test. In 2D, myotubes began to pull off the plate surface after 14 days in culture, and therefore analyses were only conducted at 7 and 14 days in 2D cultures. In 2D culture, immature (MYH 3 and 8) and slow (MYH 7) MYH relative mRNA expression was decreased at 14 days versus 7 days (p<0.05), whereas MYH 2 showed no change (0.87 to 0.98,) and MYH 1 expression showed a non-significant mean increases in expression (0.96 to 1.38, p=0.13) . In 3D, all MYH transcripts were lowly expressed at 7 days, but gradually increased in expression at 14 days before peaking significantly at 21 days (p<0.001). At 21 days in 3D culture, all MYH transcripts were significantly increased versus all other time points across conditions (p<0.001). Myotube width was significantly greater in 2D than in 3D (45.21 ± 23.62 vs 23.41 ± 4.81, p<0.001), however in 3D the myotube width was far less variable (range 10.88-101.89 in 2D vs. 15.06-34.01 in 3D). In addition, 52% of myotubes were branched in 2D compared to a complete lack of branching in 3D culture. This data demonstrates that human muscle derived cells can be cultured for longer in 3D versus 2D and at 21 days show advanced maturation based on MYH transcripts. Morphologically 3D culture is also more biomimetic than 2D, showing similar characteristics to in vivo muscle. Phenotypic changes in human skeletal muscle can now further be investigated using this model which is amenable to electrical stimulation.