AbstractAs the excitation–contraction coupling is inseparable during voluntary exercise, the relative contribution of the mechanical and neural input on hypertrophy-related molecular signalling is still poorly understood. Herein, we use a rat in-vivo strength exercise model with an electrically-induced standardized excitation pattern, previously shown to induce a load-dependent increase in myonuclear number and hypertrophy, to study acute effects of load on molecular signalling. We assessed protein abundance and specific phosphorylation of the four protein kinases FAK, mTOR, p70S6K and JNK after 2, 10 and 28 min of a low- or high-load contraction, in order to assess the effects of load, exercise duration and muscle-type on their response to exercise. Specific phosphorylation of mTOR, p70S6K and JNK was increased after 28 min of exercise under the low- and high-load protocol. Elevated phosphorylation of mTOR and JNK was detectable already after 2 and 10 min of exercise, respectively, but greatest after 28 min of exercise, and JNK phosphorylation was highly load-dependent. The abundance of all four kinases was higher in TA compared to EDL muscle, p70S6K abundance was increased after exercise in a load-independent manner, and FAK and JNK abundance was reduced after 28 min of exercise in both the exercised and control muscles. In conclusion, the current study shows that JNK activation after a single resistance exercise is load-specific, resembling the previously reported degree of myonuclear accrual and muscle hypertrophy with repetition of the exercise stimulus.