Chlamydomonas reinhardtii is able to produce molecular hydrogen in a clean way. Overall H2 release is the result of complex, interacting and transient intracellular mechanisms. To relate the dynamic coupling between culture conditions and biological responses, an original lab-scale set-up has been developed. Such device enables culture conditions to be highly controlled and provides on-line mass-spectrometric measurement of gas production. A first validation was conducted with the well-known protocol of sulfur deprivation. Biochemical analysis combined with gas flow control enabled instantaneous productivities to be calculated, and kinetic evolutions of successive physiological states to be obtained. An energetic study was also conducted. A maximal energetic yield of light conversion to H2 energy of 0.125% was achieved, far from the photosynthesis potential and usual photobioreactor efficiencies reported for biomass application (around 10%). However, the designed photobioreactor connected with data acquisition system is an innovative tool for future methodical optimization of H2 production using photosynthetic microorganisms, integrating both bioprocess and physiological aspects.