The majority of healthy, stabled horses in the absence of environmental stimuli, when managed under good welfare conditions, should exhibit relaxed and passive behaviours and physiological responses. These characteristics should be maintained during the tacking-up process without evidence of stress or anxiety. Some horses however display a wide range of abnormal postures and behaviours when being tacked up. One method in which horse owners have tried to counteract this, is the addition of a products containing Valerian, to potentially ease cases of environmentally induced anxiety. The effectiveness of these products however, is variable and require further validation. Thirty horses were involved in the current study, 10 mares and 20 castrated males of varying ages and breeds. A double-blind placebo within subjects method was used with horses being treated topically with either water or the valerian based aerosol during the tacking up process. Salivary cortisol concentrations were monitored using salivary swabs and heart rates, (HR), were monitored by measuring cardiac beat to beat intervals. In addition, eye temperature was recorded using infrared technology (IRT) and behaviours were continuously monitored. Horses at rest in the stable of pasture rarely demonstrated anxiety related behaviours (0.77% of the total time observed). After applying both aerosols during the tacking up process, these behaviours increased. There was a statistically significant difference between the physiological measures of anxiety and stress dependent on environmental stimuli in HR and eye temperature, however, there was no statistical significant difference between the salivary cortisol levels. There was a statically significant association between the environmental stimuli and the frequency of anxiety behaviours being demonstrated (p < 0.05). The valerian based product did demonstrate a reduction in heart rate in comparison to other aerosol products, in the future the mechanism of applying the calming spray must be consider due to the aversion of some individuals to aerosol application.