Upper respiratory tract infection (URTI) has a significant economic and social impact and is a major factor compromising athletes' training and competition. The effects of ColdZyme® Mouth Spray on URTI were investigated using an in vivo study in athletes, combined with a novel in vitro air-liquid interface human airway model. Endurance athletes were randomised to ColdZyme (n = 78) or placebo (n = 76) and monitored over 3 months. They completed daily symptom and training logs and collected throat swabs over 7 days during perceived URTI. In vitro studies examined rhinovirus infectivity and epithelial barrier integrity of airway epithelial cells. Eighty-two in vivo episodes were analysed with significantly lower (P = 0.012) episode duration in the ColdZyme vs. Placebo group (mean ± SD, 6.2 ± 2.6, (median [interquartile range]) 5.5 [4-9] days vs. 10.7 ± 10.2, 7.0 [5-11]). There was no difference in incidence (P = 0.149). Training absence and symptom ratings were lower (P <
0.05) in the ColdZyme group. Swabs were returned for 50 episodes, with at least one pathogen detected in all (rhinovirus was most abundant). Absolute quantification (qPCR) for rhinovirus revealed a significantly lower 7-day area under the curve in ColdZyme vs. placebo (median reduction, 94%, P = 0.029). In vitro, viral load was significantly lower (median reductions 80-100%), and epithelial barrier integrity better maintained, and no virus was detected by immunofluorescence analyses of pseudostratified epithelia, with ColdZyme treatment (all P <
0.05). ColdZyme is beneficial for reducing URTI duration, symptom ratings and missed training days. These novel data suggest that the mechanisms involve the protection of epithelial cells against rhinovirus infection and damage. KEY POINTS: Upper respiratory tract infections (URTI) are a common complaint in the general population and athletes alike, with social, well-being and economic consequences, including performance detriments in athletes and reduced work productivity in the general population. Strategies to minimise the risk of contracting a URTI and/or reduce the time taken to clear an infection are desirable to athletes and the general population alike. The present study employed an in vivo study with athletes in combination with a novel in vitro human airway cell model to examine the effects of ColdZyme Mouth Spray on URTI and viral infectivity. The duration for which URTI symptoms persisted was lower with ColdZyme treatment, which also resulted in fewer training absence days. Swabs from participants in the in vivo study and supernatants from the in vitro studies showed lower rhinovirus viral load with ColdZyme treatment compared with placebo or control.