We developed an automated microfluidic chip that can measure dynamic cytokine secretion and transcription factor activation from cells responding to time-varying stimuli. Our chip patterns antibodies, exposes cells to time-varying inputs, measures cell secretion dynamics, and quantifies secretion all in the same platform. Secretion dynamics are measured using micrometer-sized immunoassays patterned directly inside the chip. All processes are automated, so that no user input is needed for conducting a complete cycle of device preparation, cell experiments, and secretion quantification. Using this system, we simulated an immune response by exposing cells to stimuli indicative of chronic and increasing inflammation. Specifically, we quantified how macrophages respond to changing levels of the bacterial ligand LPS, in terms of transcription factor NF-κB activity and TNF cytokine secretion. The integration of assay preparation with experimental automation of our system simplifies protocols for measuring cell responses to dynamic and physiologically relevant conditions and enables simpler and more error free means of microfluidic cellular investigations.