The temperature dependent rheological and structural behavior of a long-chain C16E4 (tetraethylene glycol monohexadecyl ether) surfactant in D2O has been studied within the regime of low shear range. In the absence of shear flow, the system forms a lamellar liquid crystalline phase at relatively high temperatures. The present paper reports on the shear-induced multi-lamellar vesicle (MLV) formation in C16E4/D2O at 40 wt.% of surfactant in the temperature range of 40–55 °C. The transition from planar lamellar structure to multi-lamellar vesicles has been investigated by time-resolved experiments combining rheology and nuclear magnetic resonance (rheo-NMR), rheo small-angle neutron scattering (rheo-SANS) and rheometry. The typical transient viscosity behavior of MLV formation has been discovered at low shear rate value of 0.5 s−1.