Exercise is beneficial for several organ systems in the body and increases insulin sensitivity [1]. However, previous evidence suggests that the timing of exercise in people with Type 2 Diabetes may have opposing outcomes on glycaemia [2]. This may be particularly relevant in people also being prescribed metformin [3], the most prescribed anti-hyperglycaemic medication for people with Type 2 Diabetes and often recommended concomitantly along exercise. Although exercise improves glycaemia, this effect is inhibited when undertaken alongside metformin ingestion [4.5]. Metformin intake also appears further inhibit the beneficial response to exercise, including ablating improvements in insulin sensitivity and skeletal muscle mitochondrial protein synthesis and respiration [6.7]. Therefore, we hypothesise that the skeletal muscle signalling response to exercise is disrupted by metformin and that it may be possible to optimise recommendations for when to take metformin together with exercise. To test this, we performed a remote crossover study, 9 male and 9 females with T2D undergoing metformin monotherapy completed 2-week baseline, six weeks randomly assigned to morning (7-10am) or evening (4-7pm) exercise (30 mins), with a two-week wash-out period. Acute AUC glucose was significantly lower (p=0.01) in participants taking metformin before breakfast (152.5±29.95mmol/L) compared with participants taking metformin after breakfast (227.2±61.51mmol/L) only during the morning exercise arm. Our data indicates that morning moderate exercise lower glucose levels in people with T2D on metformin, especially when metformin is taken before breakfast. We also analysed skeletal muscle biopsies from seven healthy lean men that completed one bout of single-leg exercise with a contralateral control-leg after acute metformin/placebo supplementation [8]. After fasting overnight, participants had biopsies taken from the vastus lateralis in both legs and then received either 1.5g of metformin or a placebo with breakfast. They then rested for 4½ hours and had biopsies taken from both legs. Then, they did a 40-minute knee extensor exercise at 80%PWL followed by another biopsy. RNA sequencing analysis revealed that metformin supplementation inhibited the transcriptomic response to exercise by reducing the total number of differentially expressed genes in response to exercise. After exercise, 53 genes were upregulated in the placebo trial, while only 17 genes were upregulated in the Metformin trial. Importantly, the transcription factor NR4A3 was upregulated in response to exercise during the placebo trial (adj.p=0.011), but not when participants consumed metformin (adj.p=0.234). In Human skeletal muscle myotubes (HSMM), metformin (10µM) did not change NR4A3 expression (RT-qPCR, 1.2-fold-change, p=0.536) compared to the control. As expected, exercise mimetic Ionomycin (8µM) significantly increased NR4A3 expression (4.3-fold-change, p<0.001), however concomitant metformin incubation significantly reduced the NR4A3 response (2.4-fold-change, p=0.002). In summary, our preliminary data suggest that taking metformin before breakfast when combined with morning moderate intensity exercise lowers blood sugar compared to other treatment timings. Our mechanistic investigations reveal that NR4A3 response to exercise in skeletal muscle is inhibited by metformin, which is likely underlying the apparent inhibition of exercise-induced skeletal muscle glucose uptake with concomitant metformin. Our data suggest that it may be possible to optimised exercise timing alongside concomitant metformin to augment skeletal muscle glucose uptake.