This paper describes the development, verification and use of the first dual on-line electrochemical assay system for use with clinical microdialysis. The assay is a flow injection assay using mediated enzyme beds with electrochemical detection. The on-line assay has been specifically designed for use with head trauma patients in neurointensive care, allowing simultaneous measurement of the levels of glucose and lactate present in the dialysate with high time resolution (30 s sampling intervals). The assay is compared with the Yellow Springs 2300 glucose/lactate analyser in simple analyses and during zero net flux experiments. The on-line assay has greater accuracy and precision particularly at low concentrations of glucose and lactate. The rapid nature of this dual on-line system makes it valuable in studying the roles of energy metabolites in the brain extracellular fluid (ECF), and as an important new method for routine clinical monitoring of patients in the neurointensive care unit. Data is shown for monitoring during surgery and in intensive care.