The pathogenesis of gastrointestinal symptoms in diabetes mellitus is complex and multi-factorial. Diabetes induced peripheral and central changes in the neuronal pain matrix may be of importance and were explored using a new multi-modal and multi-segmental sensory testing approach. The sensitivity to mechanical, thermal and electrical stimulations in the oesophagus and duodenum was assessed in 12 type-1 diabetic patients with proven autonomic neuropathy and severe gastrointestinal symptoms using a comprehensive stimulation device aiming to activate different gut nerves and pain mechanisms. Twelve healthy subjects served as controls. The sensory response and the somatic referred pain areas were recorded. In the diabetic patients an overall hyposensitivity to the combination of all stimulations was found in the oesophagus and duodenum (P=0.02). Post hoc analysis revealed hyposensitivity to mechanical stimulations in the oesophagus (P=0.006) and duodenum (P=0.002), and to thermal (P<0.001) and electrical (P=0.005) stimulations in the oesophagus and duodenum combined. The hyposensitivity in the gut was accompanied by a 46% increase in the somatic referred pain areas (P=0.04) indicating central neuronal changes. The multi-modal and multi-segmental sensory testing approach indicates that the sensory nerves are widely affected in the GI tract and generalized to nerves in all layers of the gut. Changes in the neuronal pain matrix including interactions between peripheral and central pain mechanisms may be involved in the pathogenesis of gastrointestinal symptoms in long-standing diabetes. Future targets in the treatment of gastrointestinal symptoms in diabetic patients with autonomic neuropathy could be based on modulation of the central nervous system excitability.