The impact of sitting and energy imbalance on appetite and appetite-regulating hormones (acylated ghrelin and leptin) was assessed in response to 1 day of sitting, with and without changes in energy intake. Fourteen men and women completed each of three 24-h conditions: high energy expenditure (standing) with energy balance (STAND), low energy expenditure (sitting) with energy surplus (SIT), and sitting with energy balance (SIT-BAL). Ghrelin, leptin, and appetite were measured in the fasted state and following a standardized meal. In the fasted state, there were no differences among conditions. Following the meal, ghrelin was lower in SIT than in STAND, with no change in appetite. When intake was reduced (SIT-BAL), the decrease in ghrelin when sitting was attenuated, hunger increased, and fullness decreased. SIT led to lower ghrelin concentrations in the men, whereas in the women, leptin increased. SIT-BAL led to an increase in ghrelin in the men but attenuated the leptin response, reduced ghrelin, increased hunger, and decreased fullness in the women. Because a dramatic reduction in energy expenditure was not accompanied by reduced appetite, prolonged sitting may promote excess energy intake, leading to weight gain in both men and women.