Through microfluidic interrogation we analyzed real-time calcium responses of HEK293 cells stimulated with short pulses of the M3 muscarinic receptor ligand carbachol in two different concentration regimes. Lower ligand concentrations elicit oscillatory calcium signals while higher concentrations trigger a rapid rise that eventually settles down at a steady-state slightly above pre-stimulus levels, referred to as an acute signal. Cells were periodically pulsed with carbachol at these two concentration regimes using a custom-made microfluidic platform, and the resulting calcium signals were measured with a single fluorescent readout. Pulsed stimulations at these two concentration regimes resulted in multiple types of response patterns that each delivered complementary information about the M3 muscarinic receptor signaling pathway. These multiple types of calcium response patterns enabled development of a comprehensive mathematical model of multi-regime calcium signaling. The resulting model suggests that dephosphorylation of deactivated receptors is rate limiting for recovery of calcium signals in the acute regime (high ligand concentration), while calcium replenishment and IP3 production determine signal recovery in the oscillatory regime (low ligand concentration). This study not only provides mechanistic insight into multi-regime signaling of the M3 muscarinic receptor pathway, but also provides a general strategy for analyzing multi-regime pathways using only one fluorescent readout.