Substantial gains in muscle strength and hypertrophy are clearly associated with the routine performance of resistance training. What is less evident is the optimal timing of the resistance training stimulus to elicit these significant functional and structural skeletal muscle changes. Therefore, this investigation determined the impact of a single bout of resistance training performed either in the morning or evening upon acute anabolic signalling (insulin-like growth factor-binding protein-3 (IGFBP-3), myogenic index and differentiation) and catabolic processes (cortisol). Twenty-four male participants (age 21.4±1.9yrs, mass 83.7±13.7kg) with no sustained resistance training experience were allocated to a resistance exercise group (REP). Sixteen of the 24 participants were randomly selected to perform an additional non-exercising control group (CP) protocol. REP performed two bouts of resistance exercise (80% 1RM) in the morning (AM: 0800 hrs) and evening (PM: 1800 hrs), with the sessions separated by a minimum of 72 hours. Venous blood was collected immediately prior to, and 5 min after, each resistance exercise and control sessions. Serum cortisol and IGFBP-3 levels, myogenic index, myotube width, were determined at each sampling period. All data are reported as mean ± SEM, statistical significance was set at P≤0.05. As expected a significant reduction in evening cortisol concentration was observed at pre (AM: 98.4±10.5, PM: 49.8±4.4 ng/ml, P<0.001) and post (AM: 98.0±9.0, PM: 52.7±6.0 ng/ml, P<0.001) exercise. Interestingly, individual cortisol differences pre vs post exercise indicate a time-of-day effect (AM difference: -2±2.6%, PM difference: 14.0±6.7%, P = 0.03). A time-of-day related elevation in serum IGFBP-3 (AM: 3274.9 ± 345.2, PM: 3605.1 ± 367.5, p = 0.032) was also evident. Pre exercise myogenic index (AM: 8.0±0.6%, PM: 16.8±1.1%) and myotube width (AM: 48.0±3.0, PM: 71.6±1.9 μm) were significantly elevated (P<0.001) in the evening. Post exercise myogenic index was greater AM (11.5±1.6%) compared with PM (4.6±0.9%). No difference was observed in myotube width (AM: 48.5±1.5, PM: 47.8±1.8 μm) (P>0.05). Timing of resistance training regimen in the evening appears to augment some markers of hypertrophic potential, with elevated IGFBP-3, suppressed cortisol and a superior cellular environment. Further investigation, to further elucidate the time course of peak anabolic signalling in morning vs evening training conditions, are timely.