Technical Abstract: An unresolved issue in the field of diet and health is if and how changes in meal frequency affect energy metabolism in humans. We therefore evaluated the influence of reduced meal frequency without a reduction in energy intake on glucose metabolism in normal weight healthy male and female subjects. The study was a randomized cross-over design, with 2 eight-week treatment periods (with an intervening 11 week off-diet period) in which subjects consumed all of their calories for weight maintenance distributed in either 3 meals or 1 meal per day (consumed between 17:00 and 21:00). Energy metabolism was evaluated at designated time points throughout the study by performing morning oral glucose tolerance tests (OGTT) and measuring levels of glucose, insulin, glucagon, leptin, ghrelin, adiponectin, resistin and brain-derived neurotrophic factor (BDNF). Subjects consuming 1 meal/d exhibited higher morning fasting plasma glucose levels, greater and more sustained elevations of plasma glucose concentrations and a delayed insulin response in the OGTT compared to subjects consuming 3 meal/d. Levels of ghrelin were elevated in response to the 1 meal/d regimen. Fasting levels of insulin, leptin, ghrelin, adiponectin, resistin and BDNF were not significantly affected by meal frequency. Subjects consuming a single large daily meal exhibit elevated fasting glucose levels, and impaired morning glucose tolerance associated with a delayed insulin response, during a 2 month diet period compared to those consuming 3 meals/day. The impaired glucose tolerance was not associated with alterations in the levels of adipokines or BDNF.