Food-restriction increases hunger and reduces thermogenesis, which are homeostatic mechanisms to defend against weight loss. A primary driver of this metabolic response is increased neuropeptide Y (NPY) and agouti-related protein (AgRP) expression in the arcuate nucleus (ARC). In sheep, exercise counteracts the impaired thermogenesis in brown adipose tissue (BAT) caused by food-restriction. Despite this, exercise does not change the expression of markers for BAT activity or the ‘browning’ of white adipose tissue. Appetite-regulating peptides exert dual control over food intake and thermogenesis and thus the protective effect of exercise may manifest at this level. Our aim was to characterise the effects of exercise and/or food-restriction (diet) on the expression of hypothalamic appetite-regulating peptides in anestrous ewes. Normal weight animals were randomly divided into four groups (n=5), including control sedentary fed ad lib, exercise fed ad lib (30 min/ day, 5 days/ week) diet-restricted (70% of ad lib food intake) and combined diet and exercise. Interventions were carried out for 4 weeks after which, sheep were euthanised and hypothalami perfused for in situ hybridisation analyses. Diet and exercise combined reduced (<0.001) adiposity, with no singular effect of either intervention. NPY but not AgRP gene expression/cell increased (P<0.05) with diet restriction and overall expression of NPY and AgRP gene expression increased (P<0.05) with combined diet/exercise, consistent with reduced adiposity. Neither diet nor exercise affected pro-opiomelanocortin expression in the ARC, but diet increased (P<0.01) expression of the melanocortin 4 receptor (MC4R) in the paraventricular nucleus, irrespective of exercise. In the lateral hypothalamus, exercise alone increased (P<0.01) orexin expression, an effect abolished by food restriction. In conclusion, combined diet and exercise reduced adiposity, which was associated with increased expression of orexigenic peptides. The effect of diet to counteract increased expression of orexin in exercising animals may be a protective mechanism to reduce energy expenditure.