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The aim of this study was to evaluate the influence of shearing and road transport on reactive oxygen species (dROMs), antioxidant barrier (Oxy-ads), and thiol antioxidant barrier (SHp) concentrations in healthy Comisana ewes. Thirty animals were divided into 3 equal groups: group A was used as the control, group B was shorn, and group C was transported by road for 6 hours. For animals in experimental groups (groups B and C), blood samples were collected before (T0) and immediately after (T1) the shearing and/or road transport procedures and 24 (T2) and 48 (T3) hours from the end of each procedure. For the control group, blood sampling was performed for the same time/data points as for the experimental groups. A 2-way repeated-measures analysis of variance showed significant higher dROMs, Oxy-ads, and SHp values (P < 0.05) in group B compared to control group at T3. Group C showed significant higher dROMs, Oxy-ads, and SHp values (P < 0.05) than control group at T1, T2, and T3. The results obtained in this study suggest that road transport mostly affected the oxidant/antioxidant status in ewes. In addition, the trend of dROMs values together with trend of Oxy-Ads and SHp values found in our study suggest that despite the increment of oxidative phenomena occurring following shearing and transport, oxidative homeostasis was maintained by the efficiency of the antioxidant defenses both in shorn and transported ewes.