This protocol describes the design, fabrication and use of a 3D physiological and pathophysiological motor unit model consisting of motor neurons coupled to skeletal muscles interacting via the neuromuscular junction (NMJ) within a microfluidic device. This model facilitates imaging and quantitative functional assessment. The 'NMJ chip' enables real-time, live imaging of axonal outgrowth, NMJ formation and muscle maturation, as well as synchronization of motor neuron activity and muscle contraction under optogenetic control for the study of normal physiological events. The proposed protocol takes ~2-3 months to be implemented. Pathological behaviors associated with various neuromuscular diseases, such as regression of motor neuron axons, motor neuron death, and muscle degradation and atrophy can also be recapitulated in this system. Disease models can be created by the use of patient-derived induced pluripotent stem cells to generate both the motor neurons and skeletal muscle cells used. This is demonstrated by the use of cells from a patient with sporadic amyotrophic lateral sclerosis but can be applied more generally to models of neuromuscular disease, such as spinal muscular atrophy, NMJ disorder and muscular dystrophy. Models such as this hold considerable potential for applications in precision medicine, drug screening and disease risk assessment.