Type 2 Diabetes Mellitus (T2D) is a growing global problem. Mutations in core-clock genes increase the risk of developing T2D1, and a disrupted core-clock leads to obesity and insulin resistance in rodent models. Defective mitochondrial metabolism within skeletal muscle appears to play a major role in insulin resistance2, and mitochondrial membrane dynamics may be an important dysregulated pathway in T2D3,4. We sought to test the hypothesis that there are intrinsic rhythmic differences in skeletal muscle from people with T2D, and that this is linked to dysfunctional mitochondrial metabolism. Synchronised human skeletal muscle cells were harvested every 6 hours over 42 hours and underwent RNA-seq. The core-clock genes CLOCK, CRY1, NPAS2, NR1D1 and PER3 displayed different cycling patterns in myotubes from normal glucose tolerant controls (NGT) compared to T2D (p<0.05, determined via ‘DODR’ analysis). Myotubes from NGT had circadian rhythm in oxygen consumption rate oscillation (p<0.05, RAIN), whereas T2D did not (both n=5). Myotubes from NGT’s displayed more cycling genes in the mitochondrial inner membrane than T2D (Gene ratio T2D:>0.11; NGT:>0.26, adjusted p-value for compartment enrichment T2D:<0.01; NGT:<0.05). Vastus lateralis muscle from NGT (n=25) and T2D (n=25) volunteers was obtained using a needle biopsy technique. Microarrays were performed on all samples. These subjects also underwent a euglycemic-hyperinsulinemic clamp to determine an M-value for insulin sensitivity. Cycling genes specific to NGT from the inner-mitochondrial membrane (analysed as a pathway) positively correlated with M-value (correlation: q<0.05), including inner-mitochondrial membrane regulator OPA1. Skeletal muscle specific OPA1-/- mice displayed altered gene expression of core-clock genes: Clock (p<0.001:t-test, OPA1-/- 0.81±0.05:SEM vs Wild-type 1.21±0.11:SEM), Bmal1 (p<0.05:t-test, OPA1-/- 0.68±0.28:SEM vs Wild-type 1.37±0.11:SEM) and Nr1d1 (p<0.001:t-test, OPA1-/- 0.63±0.07:SEM vs Wild-type 1.41±0.09:SEM). siRNA targeting OPA1 in synchronised primary human skeletal myotubes altered expression of NPAS2 (a paralogue of Clock) and PER2 (both genes: 2-way ANOVA: p<0.05 time-interaction; siOPA1 vs scramble). OPA1 depletion also resulted in an altered rhythmic oxygen consumption rate (12hrs siOPA1 vs 4hrs Scramble: p<0.01). These are the first data to demonstrate intrinsically dysregulated core-clock rhythmicity in primary cells from patients with T2D. Mitochondrial dynamics have previously been implicated in circadian regulation of the core-clock, in this study we also show that OPA1 expression in skeletal muscle is positively correlated with insulin sensitivity. OPA1 expression oscillates in a circadian manner in primate skeletal muscle5, and these data demonstrate disruption of OPA1 in skeletal muscle in vivo. and in vitro alters retrograde signalling from the inner-mitochondrial membrane that affects the core-clock in skeletal muscle.