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Aim: The acute myocellular responses of caffeine supplementation during resistance exercise (RE) have not been investigated. β2-Adrenergic receptors (β2AR) may be a target of the stimulatory effects of caffeine and stimulate bioenergetic pathways including protein kinase A (PKA), and mitogen-activated protein kinases (MAPK). Purpose: Elucidate the effects of pre-workout supplementation on signaling responses to an acute RE bout. Methods: In a randomized, counter-balanced, double-blind, placebo-controlled, within-subject crossover study, ten resistance-trained males (mean ± SD; age = 22 ± 2.4 years, height = 175 ± 7 cm, body mass = 84.1 ± 11.8 kg) consumed a caffeine containing multi-ingredient pre-workout supplement (SUPP) or color and flavor matched placebo (PL) 60 min prior to an acute RE bout of barbell back squats. Pre- and post-exercise muscle biopsies were analyzed for the phosphorylation (p-) of β2AR, PKA, and MAPK (ERK, JNK, p38). Epinephrine was determined prior to supplementation (baseline; BL), after supplementation but prior to RE (PRE), and immediately after RE (POST). Results: Epinephrine increased at PRE in SUPP (mean ± SE: 323 ± 34 vs 457 ± 68 pmol/l; p = 0.028), and was greatest at POST in the SUPP condition compared to PL (5140 ± 852 vs 2862 ± 498 pmol/l; p = 0.006). p-β2AR and p-MAPK increased post-exercise (p < 0.05) with no differences between conditions (p > 0.05). Pearson correlations indicated there was a relationship between epinephrine and p-β2AR in PL (r = − 0.810; p = 0.008), and p-β2AR and ERK in SUPP (r = 0.941; p < 0.001). Conclusion: Consumption of a caffeine containing pre-workout supplement improves performance, possibly through increases in pre-exercise catecholamines. However, the acute myocellular signaling responses were largely similar post-exercise. [ABSTRACT FROM AUTHOR]