There is increasing evidence for glucose fluctuation playing a role in the damaging effects of diabetes on various organs, including the brain. We aimed to study the effects of glycaemic variation (GV) upon mitochondrial activity using an in vitro human neuronal model. The metabolic disturbance of GV in neuronal cells, was mimicked via exposure of neuroblastoma cells SH-SY5Y to constant glucose or fluctuating (i.e. 6 h cycles) for 24 and 48 h. Mitochondrial dehydrogenase activity was determined via MTT assay. Cell mitochondrial activity (MTT) was moderately decreased in constant high glucose, but markedly decreased following 24 and 48 h of cyclical glucose fluctuations. Glucose transport determined via 2-deoxy-D-[1-(14)C] glucose uptake was regulated in an exaggerated manner in response to glucose variance, accompanied by modest changes in GLUT 1 mRNA abundance. Osmotic components of these glucose effects were investigated in the presence of the osmotic-mimics mannitol and L: -glucose. Both treatments showed that fluctuating osmolality did not result in a significant change in mitochondrial activity and had no effects on (14)Cglucose uptake, suggesting that adverse effects on mitochondrial function were specifically related to metabolically active glucose fluctuations. Apoptosis gene expression showed that both intrinsic and extrinsic apoptotic pathways were modulated by glucose variance, with two major response clusters corresponding to (i) glucose stress-modulated genes, (ii) glucose mediated osmotic stress-modulated genes. Gene clustering analysis by STRING showed that most of the glucose stress-modulated genes were components of the intrinsic/mitochondrial apoptotic pathway including Bcl-2, Caspases and apoptosis executors. On the other hand the glucose mediated osmotic stress-modulated genes were mostly within the extrinsic apoptotic pathway, including TNF receptor and their ligands and adaptors/activators/initiators of apoptosis. Fluctuating glucose levels have a greater adverse effect on neuronal cell energy regulation mechanisms than either sustained high or low glucose levels.