Introduction: Along with climate changes, we see an increase in allergic symptoms and the number of pollen-allergic patients in many countries. Increased allergic symptoms are associated with an elevated ozone exposure which may be linked by impaired epithelial barrier function. This study aimed to quantify the clinical effect of ozone and pollen double exposure (DE). We tested whether ozone impairs barrier-related skin physiology and mucosal functions under double exposure with pollen ozone in grass pollen-allergic patients versus healthy controls. Methods: This case-control study included 8 grass pollen-allergic patients and 8 non-allergic healthy subjects exposed to grass pollen and ozone in the GA(2)LEN pollen chamber, comparing shorter and longer DE duration. Non-invasive skin physiological parameters were assessed, including stratum corneum hydration, skin redness, surface pH, and basal transepidermal water loss (TEWL) as a parameter for epidermal barrier function. The subjects' general well-being, bronchial, nasal, and ocular symptoms were documented. Results: Skin physiology tests revealed that DE in allergic patients deteriorates the epidermal barrier function, increases the surface pH and skin redness. DE significantly induced nasal secretion in pollen-allergic versus healthy subjects, which was more pronounced with longer DE. The general well-being was significantly impaired under DE versus pollen or ozone alone, with a negative influence of DE-duration. No relevant bronchial symptoms were recorded. Conclusion: Skin physiology and nasal mucosal symptoms and are negatively affected by ozone and grass pollen DE in allergic patients. The negative effects showed, in some parameters, a dose(time)-response relationship. The surface pH can be regarded as a possible modulatory mechanism.