Increased skin pH and transepidermal water loss (TEWL) are documented in atopic people and dogs but no study has investigated how these parameters change during an allergy flare. Our primary aim was to challenge atopic beagles to dust mites and measure pH and TEWL during a flare of atopic dermatitis and correlate these parameters to clinical signs. A secondary aim was to evaluate in a randomized placebo-controlled study whether the daily application of 50/50 vinegar spray improves clinical signs and affects skin parameters despite the allergen challenge. Fifteen atopic dogs were challenged epicutaneously twice weekly for 2 weeks with allergen application on the inguinal and medial thigh areas. The severity of dermatitis was scored daily (CADESI-03). TEWL and pH were measured daily on the inguinal and medial thigh areas. A repeated measures ANOVA showed the significant effect of time, with increased pH (p < 0.0001), TEWL (p < 0.0001), and CADESI (p < 0.0001) during allergen challenge. Significant positive correlations were found between CADESI and pH (r = 0.3556; p < 0.0001), CADESI and TEWL (r = 0.36; p < 0.0001), and pH and TEWL (r = 0.45; p < 0.0001). Daily application of 50/50 vinegar did not improve dermatitis, pH, and TEWL compared to the control treatment. It can be concluded that both pH and TEWL are markers of disease severity in canine atopic dermatitis.