We examined the effects of eating at night on levels of triglyceride (TG) in healthy young adults. We find, that even in this population, eating at night results in higher circulating TG levels compared to eating calorically and nutritionally equivalent meals during the day. When eating hourly isocaloric test meals across 40 consecutive hours, circulating TG levels were high when meals were consumed in the early morning hours, peaking around 03:30 h, compared to eating the same meals during day. This continuous feeding pattern enabled us to identify the most ‘vulnerable’ time for eating with respect to TG levels which will be important to help guide healthy eating decisions in individuals who by choice, or necessity (e.g., shift workers) are awake and eating at these times. We also found that meal size was an important factor when assessing the time-of-day response to food intake: When eating at night, only half the calories were needed to reach daytime TG levels. These data show that, not only does eating at night lead to higher circulating levels of TG compared to the day, but that these levels are achieved having eaten less food. The adverse changes in circulating lipid profiles associated with nighttime eating reported in these healthy young adults may have important implications for the etiology of dyslipidemia in shift workers who are chronically eating during the night. Our study also examined circadian rhythms in lipid profiles. By comparing daily patterns in lipid levels between a standard daytime meal schedule and meals consumed around the clock, we demonstrated significant disruption in the temporal organization of lipid metabolism due to eating patterns. We found that when eating on a normal schedule, all lipids (total cholesterol, TG, LDL-C, HDL-C, VLDL-C) were in alignment and peaked during the afternoon (16:00 h). When eating occurred during both the day and night, however, this alignment broke down such that TG and VLDL-C peaked at night, 10 hours later than normal, and 10 hours later than the other lipids that remained unchanged. The consequences of this internal misalignment in lipid metabolism are as yet unknown but it may contribute to the development of dyslipidemia and cardiovascular disease in shift workers.