Alterations in metabolism and heart rate variability (HRV) and endothelial dysfunction as well as sleep deprivation and shift work have been associated with an increased risk for cardiovascular disease. The aim of the current study was to investigate the effect of one night of total sleep deprivation (TSD) (as a proxy for the first night of shift work) and recovery sleep on metabolic and cardiovascular function and alertness and mood under controlled laboratory conditions in experienced shift workers compared with non-shift workers. Eleven shift workers (SW) (shift work > 5 years) and 14 non-shift workers (NSW), matched for age, BMI and cholesterol, followed a 7-day regular sleep-wake cycle prior to the laboratory session, consisting of adaptation sleep, baseline sleep, TSD (30.5 h), a 4-h nap and recovery sleep. All interventions were relative to wake up time (body posture, food intake and light controlled throughout). After TSD, basal plasma triacylglycerol (TAG) levels and postprandial glucose clearance decreased and the morning peak in HRV variance increased. After recovery sleep, TAG and insulin responses increased and non-esterified fatty acid (NEFA) responses and HRV variance decreased. These data suggest shifts in insulin sensitivity and sympathetic/parasympathetic balance after TSD and recovery sleep. Mood and alertness deteriorated after TSD but the recovery periods provided effective recuperation. There were no differences in screening parameters and circadian phase between SW and NSW. However, SW felt more alert, cheerful, elated and calmer and showed a lower HRV variance and higher sympathetic activity as well as a trend for lower endothelial function throughout the study and a smaller increase in insulin and decrease in NEFA responses after recovery sleep. Further research is needed to unravel the mechanisms underlying these group differences and to clarify why some changes in metabolic and cardiovascular function take place after TSD while others are observed following recovery sleep.