Cortisol is often used as an indicator of physiological stress in animals. Although traditional sampling methods (blood, saliva, urine, feces) are utilized to measure acute cortisol secretion, hair cortisol analysis is a non-invasive method to quantify long-term cortisol secretion in mammals. Research has shown that a variety of environmental factors, including housing conditions, can impact horse health, behavior, and performance. The objectives of this study were to 1) evaluate the influence of transitioning horses from outdoor group housing to individual box stalls on hair cortisol concentrations (HCC) and 2) determine the correlation between HCC, heart rate (HR) and behavior during novel object tests. Hair samples were collected approximately every 30d from the same location of 11 Quarter Horses (5 mares, 6 geldings; 3.69 + 1.4yr) over a period of 5 months. Cortisol was extracted into methanol from ground hair samples and measured via ELISA. HR and behavior were recorded throughout the novel object tests. Data were analyzed using PROC MIXED in SAS v. 9.4. Overall, there was no difference in HCC due to housing conditions. However, HCC did increase during the 30d period immediately following the housing transition (p<0.05), then decreased by the following 30d period. HCC was negatively correlated with HR during the novel object tests (r2=-0.2; p<0.05). The correlation between HCC and behavior was dependent on the novel object. The use of hair cortisol analysis has the potential to provide horse owners and caretakers with a non-invasive tool to monitor long-term stress in response to management practices.