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Although skin health is of critical importance for all horse owners, understanding of cutaneous pH and trans-epidermal water loss (TEWL) and the normal ranges of these variables is lacking. Cutaneous pH and TEWL measures provide insight into the barrier function of the epidermis and have the potential to determine the resident dermal microbiota. However, these measures have been recorded to fluctuate due to a variety of factors such as environment and anatomical locations in other species. Accordingly, the objectives of this study were to identify the impacts of anatomical location, weather, and time of day on cutaneous pH and TEWL in equines. This study utilized nine (n = 9) stock type mares, group-housed in a pasture setting at Southern Illinois University Equine Center. Researchers measured cutaneous pH and TEWL three times (6:00 AM, 12:00 PM, 6:00 PM) daily across five anatomical locations (nose, whither, heart-girth, mid-back, and base of tail) for five consecutive days. All statistical analysis was conducted using SAS On Demand for Academics (P < 0.05). Analysis of changes in variables associated with time, anatomical location, and day were conducted utilizing a PROC GLM three-way repeated measures ANOVA utilizing a Greenhouse-Geiser correction. Cutaneous pH results displayed significant main effects of day (P = 0.0052) and time (P = 0.0073) as well as a significant interaction of day and location (P = 0.0004). Mean cutaneous pH fluctuated daily, however, certain anatomical locations fluctuated more than others each day. Additionally, pH was highest in the morning (mean of 7.28) and lowest in the evening (mean 7.011). However, the mean overall pH for the study is 7.14 which is congruent with other work. TEWL measurements had significant main effects of day (P < 0.0001), time (P < 0.0001), and location (P = 0.0231), as well as a significant interaction of day, time, and location (P < 0.0001). TEWL measurements fluctuated with day, likely a result of weather changes. Mean TEWL measurements were less in the morning (mean 24.68) and greatest in the afternoons (mean 38.52). Overall mean TEWL measurement was 30.25. The results of this work indicate fluctuations in dermal barrier variables are associated with day, anatomical location, and time of day. These findings may have implications for applications of dermal treatments for horses as well as catalog the natural fluctuations which may affect future research. [ABSTRACT FROM AUTHOR]