Misalignment of the daily sleep-wake and fasting-feeding cycles with the endogenous circadian timing system is an inevitable consequence of night shift work and is associated with adverse metabolic health effects. However, a detailed characterisation of the effects of night shifts on 24-h rhythms in the metabolome is missing. We performed targeted metabolomic profiling on plasma samples collected every 2 h from healthy human subjects during two 24-h measurement periods at baseline and on the fourth day of a simulated night shift protocol, in which the habitual sleep-wake cycle was delayed by 10 h. Thirty-two out of the 130 detected metabolites showed a 24-h rhythm both at baseline and during the night shift condition. Among these, 75% were driven by sleep-wake and fasting-feeding cycles rather than by the endogenous circadian clock, showing an average phase delay of 8.8 h during the night shift condition. Hence, the majority of rhythmic metabolites were misaligned relative to the endogenous circadian system during the night shift condition. This could be a key mechanism involved in the increased prevalence of adverse metabolic health effects observed in shift workers. On the individual level, the response to the night shift protocol was highly diverse, with phase shifts of rhythmic metabolite profiles ranging from a 0.2-h advance in one subject to a 12-h delay in another subject, revealing an individual metabolomic signature of circadian misalignment. Our findings provide insight into the overall and individual responses of the metabolome to circadian misalignment associated with night schedules and may thereby contribute to the development of individually tailored strategies to minimise the metabolic impacts of shift work.