Poor postprandial glucose control is a risk factor for multiple health conditions. The second-meal effect refers to the progressively improved glycaemic control with repeated feedings, an effect which is achievable with protein ingestion at the initial eating occasion. The most pronounced glycaemic response each day therefore typically occurs following breakfast, so this study investigated whether ingesting protein during the night could improve glucose control at the first meal of the day. In a randomised cross-over design, fifteen adults (7 males, 8 females; age, 22 ± 3 years; BMI, 24.0 ± 2.8 kg·m-2; fasting blood glucose, 4.9 ± 0.5 mmol·L-1) woke at 0400 ± 1 h to ingest 300 ml water with or without 63 g whey protein. Participants then completed a mixed-macronutrient meal tolerance test (1 g carbohydrate·kg body mass-1, 563 ± 104 kcal,) 5 h 39 min following the nocturnal feeding. Nocturnal protein ingestion increased the glycaemic response (incremental area under curve) to breakfast by 43.5 ± 55.5 mmol•120 min•L-1 (p=0.009, d=0.94). Consistent with this effect, individual peak blood glucose concentrations were 0.6 ± 1.0 mmol·L-1 higher following breakfast when protein had been ingested (p=0.049, d=0.50). Immediately prior to breakfast, rates of lipid oxidation were 0.02 ± 0.03 g·min-1 higher (p=0.045) in the protein condition, followed by an elevated postprandial energy expenditure (0.09 ± 0.12 kcal·min-1, p=0.018). Postprandial appetite and energy intake were similar between conditions. This study reveals a paradoxical second-meal phenomenon whereby nocturnal whey protein feeding impaired subsequent glucose tolerance, whilst increasing postprandial energy expenditure.