Water-driven self-assembly of lipids displays a variety of liquid crystalline phases that are crucial for membrane functions. In this work, we characterized the temperature-induced phase transitions in aqueous self-assembly systems using steady-state and time-resolved fluorescence measurements. The polar head group region was investigated using tryptophan (Trp) and two of its ester derivatives, and the hydrophobic tail region was probed using pyrene. The spectral changes in tryptophan and pyrene were used as a benchmark to estimate the polarity of the head group region and the tail region, respectively. A basic medium was detected and estimated for the polar region in the inverse cubic phase of a Guerbet glycolipid and was attributed to the structural effect of the narrow nanochannels. All the studied lipid compositions show completely reversible temperature-induced phase transitions, reflecting the thermodynamic equilibrium structures of their mesophases. The results reveal a large degree of heterogeneity and flexibility of the lipid self-assembly which may be crucial for carrying out different biological functions.