The ability to evaluate the extent of poor welfare during clinical disease is of great importance for animal welfare. Chronic exposure to stressful situations has a negative impact on animal welfare, and there is a need for valid and reliable biomarkers of chronic stress. The aim of this study was 1) to evaluate hair cortisol (HC) and hair cortisone (HCn) as measures of chronic stress in sheep and 2) to assess stress and pain associated with ovine footrot using different physiological [faecal cortisol metabolites (FCM), HC and HCn] and behavioural measures. The study was performed as a single foot inoculation using a boot. The right hind foot of 24 lambs was inoculated with different strains of D. nodosus, and the animals were subsequently monitored daily for two weeks to assess lameness and pain. Hair was collected from both hind limbs at the start of the study and before treatment with gamithromycin subsequent to the trial period, and faecal samples were collected weekly for four weeks. Clinical signs of footrot developed in all experimental groups. There was an increase in FCM from week 0 to 2 (p<0.001), and then a tendency of a subsequent decrease from week 2 to 3 (p=0.06), indicating a chronic stress response due to the maceration caused by the boot and the developing infection. FCM decreased to baseline levels after the animals were treated with gamithromycin. Surprisingly, the concentration of HC decreased from week 0 to week 3 (p<0.001) in the right and left limb, and significantly more in the right limb (p<0.01). The concentration of HCn increased from week 0 to week 3 (p=0.05) in the right limb but decreased in the left limb (p<0.05). Hence, our findings suggest local production and/or metabolism of glucocorticoids in the hair follicles, which should be taken into consideration in studies using HC as a parameter of chronic stress. This study provides a first indication for a potential merit of hair cortisone as a biomarker of stress in sheep.