Neuronal innervation regulates the secretion of neurotrophic myokines and exosomes from skeletal muscle. Skeletal muscles, which are responsible for voluntary movement and homeostasis, secrete myokines and exosomes that influence brain function. This study shows that neuronal innervation and activity significantly regulate the secretion of neurotrophic myokines and exosomes from skeletal muscle. Using an engineered neuromuscular tissue model, the researchers found that innervated muscles secreted higher levels of irisin and exosomes containing more diverse neurotrophic microRNAs compared to neuron-free muscles. These muscle-derived factors enhanced the growth and activity of hippocampal neurons. The study highlights the importance of neuronal innervation in modulating muscle-derived factors that support neuronal function and suggests that the engineered neuromuscular tissue model is a promising platform for producing neurotrophic molecules. The results indicate that neuronal innervation plays a key role in regulating muscle metabolism, promoting the expression of neurotrophic myokine-encoding mRNAs, and increasing the secretion yield and neurotrophic activity of myokines and exosomes. The study also reveals that neuronal innervation and firing influence the secretion of exosomes carrying miRNA cargos involved in nervous system development. The findings suggest that the neuromuscular model can be used to investigate how muscles regulate exosome packaging and release in response to neuronal signaling. The study provides insights into the role of neuronal innervation in modulating muscle secretion and its implications for neuromuscular diseases.Neuronal innervation regulates the secretion of neurotrophic myokines and exosomes from skeletal muscle. Skeletal muscles, which are responsible for voluntary movement and homeostasis, secrete myokines and exosomes that influence brain function. This study shows that neuronal innervation and activity significantly regulate the secretion of neurotrophic myokines and exosomes from skeletal muscle. Using an engineered neuromuscular tissue model, the researchers found that innervated muscles secreted higher levels of irisin and exosomes containing more diverse neurotrophic microRNAs compared to neuron-free muscles. These muscle-derived factors enhanced the growth and activity of hippocampal neurons. The study highlights the importance of neuronal innervation in modulating muscle-derived factors that support neuronal function and suggests that the engineered neuromuscular tissue model is a promising platform for producing neurotrophic molecules. The results indicate that neuronal innervation plays a key role in regulating muscle metabolism, promoting the expression of neurotrophic myokine-encoding mRNAs, and increasing the secretion yield and neurotrophic activity of myokines and exosomes. The study also reveals that neuronal innervation and firing influence the secretion of exosomes carrying miRNA cargos involved in nervous system development. The findings suggest that the neuromuscular model can be used to investigate how muscles regulate exosome packaging and release in response to neuronal signaling. The study provides insights into the role of neuronal innervation in modulating muscle secretion and its implications for neuromuscular diseases.