Abstract Objectives The neurotransmitters γ-aminobutyric acid (GABA) and glutamic acid are present in many common components of our diet like for example tomato. Recent research suggests that oral supplementation of especially GABA can have relevant health effects with regard to diabetes and blood pressure. As of yet it is unclear whether these neurotransmitters are also bioavailable from a food matrix. In this controlled cross-over human intervention study, we aimed to evaluate the relative bioavailability of GABA and glutamic acid from tomato. Methods Eleven healthy men received one liter of water containing 888 mg/L GABA or 3673 mg/L glutamic acid, a liter blended tomato juice or a liter water. Solutions were given one week apart in a random order in a four way crossover design. On every test day 19 blood samples were collected in a period of 24 hours after ingestion of the test product. Plasma GABA and glutamic acid concentrations were determined with ultra-pressure liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Results Baseline plasma GABA and glutamic acid concentrations were found to be 16.71 (SD 2.18) ng/mL and 4625.6 (SD 1666.1) ng/mL respectively. GABA baseline levels were constant (5.8 CV%) within individuals over the four consecutive test days while glutamic acid baseline levels varied considerably (23.5 CV%). Interestingly, GABA was determined to be just as bioavailable from tomato juice as from a solution in water; kinetic parameters (AUC, Cmax, Tmax) were not significantly different. For glutamic acid, the tomato food matrix did significantly alter the plasma kinetics, the maximum concentration was lower and the time to the maximum concentration was longer in the tomato condition as compared to glutamic acid dissolved in water. Conclusions These data suggest that GABA is bioavailable from food, and that food products containing GABA could potentially induce health effects similar to what is described for GABA supplements. Future research should focus on determining the bioavailability of GABA from other food products as well as the health effects of a GABA rich diet in humans. Funding Sources This work was supported by a consortium of companies (Agrico Research, Nunhems Netherlands, AVEBE) taking part in a collaborative public-partnership under the “Topsector Agri-Food” programme organised by the Dutch Ministry of Economic Affairs.