Objectives To assess effects of dehydration on tooth colour. To investigate any change in tooth colour resulting from dehydration and the time required for any change to return to baseline. Methods 20 subjects with intact maxillary central incisors were recruited. In each case one incisor was randomly assigned as test tooth with the other acting as control. Spectrophotometric shade of the test teeth was assessed before dehydration and after rubber dam isolation every 10 min for 30 min. Test teeth were allowed to rehydrate and measurements taken every 10 min for 30 min. Data was collected in CIE L*a*b* colour coordinates. A panel of 10 examiners assessed before and after dehydration digital images of the control and test teeth. The panel judged whether central incisors were of same or different shade and which tooth was lighter if a difference was detected. Results All colour coordinates showed significant differences between baseline versus 10, 30 min of dehydration and 30 min of rehydration (p < 0.02) except L* after 30 min rehydration. The panel found test and control teeth to be of same shade before and of different shade after dehydration (p < 0.001). Test teeth were significantly lighter after dehydration (p < 0.005). Conclusion A significant change in shade of teeth when dehydrated was detected by both instrumental and visual assessment. Teeth became lighter and perceivable colour change had not returned to baseline shade within 30 min of rehydration. Therefore shade matching procedures should be carried out before the teeth are exposed to dehydration. Clinical significance As most dental procedures lead to dehydration of teeth which can alter their shade and may lead to errors in shade matching. To avoid unacceptable mismatch of colour between natural teeth and dental restoration it is important that the shade matching procedure is carried out at the beginning of the appointment.