Can a water-based mist form of the F3 facial pheromone have a short-term effect on cats’ adaptation to a new situation?

Abstract

Objectives

The arrival of an unknown human in the life of a cat can be seen as a challenging situation. Helping cats cope with such situations may help during adoption processes or when introducing a pet sitter. This study aimed to investigate the impact of a water-based mist formulation of an F3 analogue on the behaviour of cats exposed to the presence of an unfamiliar human.

Methods

Two parallel and homogeneous groups of 11 cats were included in this study. Each cat was brought individually to an unknown room previously sprayed with the F3 pheromone analogue mist or a placebo. After 40 mins, an unfamiliar human entered the room and the cat’s behaviour was observed. Locomotion, rubbing, feeding and behaviours directed towards the human were analysed.

Results

F3-treated cats exhibited significantly more rubbing than those in the control group (generalised linear mixed model [GsLMM], χ² = 14.056, degrees of freedom [DF] = 1; P <0.001). They also sat (GsLMM, χ² = 6.058, DF = 1; P <0.05) and moved (GsLMM, χ² = 6.163, DF = 1; P <0.05) more frequently than the control cats. No statistically significant effects were detected for human-directed behaviours, although cats in the F3-treated room tended to be quicker to approach (frailty model, χ² = 3.54, DF = 1; P = 0.06) and initiate contact with the unknown human (frailty model, χ² = 3.454, DF = 1; P = 0.063). An exploratory principal component analysis suggested that F3-treated cats were more homogeneous in their behaviour than control cats and that these F3 cats could display a more confident behavioural profile, as assessed by their exploratory behaviour.

Conclusions and relevance

These results highlight the interest in using an F3 analogue treatment in a mist form to help cats cope with situations involving the first encounter with a new human. This was achieved by improving the approach with a new context, exploration and human interactions.

Keywords

Pheromone coping novelty human–animal relationship

Introduction

Chemical signals are known to play a crucial role in cat communication and can be used to facilitate or help prevent the display of certain species-specific behaviours.^1
–5 Among those chemicals, in mammals, pheromones are defined as chemicals emitted by an individual into the surrounding environment where, when detected by the vomeronasal organ of an individual of the same species, they will induce consistent behavioural and/or physiological changes.^6,7 These pheromones are secreted by glands located in various parts of the cat’s body, including the skin around the mammary glands, head, digits and perianal area.^8
–11 Five distinct feline facial pheromones (FFPs) that are secreted on the head or cheeks have been identified.^2,12 Although the role of two of these fractions, F1 and F5, remains unknown, the functions of F2, F3 and F4 have been established.² F2 is primarily involved in sexual behaviour,² whereas F4 is associated with social interactions, such as allorubbing.² The F3 pheromone, which is deposited when cats rub against objects, is believed to enhance their sense of security and reduce distress when faced with challenging situations.^2,3,9,13

Domestic cats will have to face many challenging situations across their life: a new environment, transportation, veterinary visits, social conflicts, chronic diseases and the arrival of a new member in the family, animal or human.¹⁴ In all these cases, the presence of chemical communication plays a crucial role in facilitating the adaptation process.

Indeed, compared with when they are in the presence of their owner, cats will explore and rub less when with a stranger.¹⁵ They will also take more time to approach the human and reduce their physical contact with them.¹⁵ These findings highlight that cats may feel less at ease in their environment when exposed to an unfamiliar human. In addition to a new human, a new environment can also be challenging for a cat. Indeed, compared with dogs, cats show a lower success rate in an open field-like habituation task.¹⁶ These findings underscore the importance of developing effective strategies to help cats adapt to new situations. Drugs could be used to help the cat to cope with the situation, but they can have side effects. Finding non-pharmacological methods to promote adaptation in cats without risking side effects is a growing area of interest in feline behaviour and welfare research.

For years now, pheromonal treatments have been used to alleviate the physiological and behavioural consequences of anxiogenic situations.^1,2 The use of F3 analogues has been particularly well documented. Hence, in one study, researchers observed that using an F3 analogue in a spray form (FELIWAY Classic Spray; Ceva Santé Animale) reduced the cat’s stress score, based on its behaviour.¹⁷ Other forms of F3 application have shown beneficial effects on cats, such as an electric diffuser (FELIWAY Classic Diffuser; Ceva Santé Animale); indeed, in a first-of-its-kind study, the diffuser was shown to increase resting behaviours and reduce stress, as evidenced by a decrease in sneezing frequency in kittens suffering from herpesvirus-1 infection,¹⁸ while, when used for 35 days, it reduced cortisol secretion in 75% of the 28 cats monitored in another study.¹⁹ As these earlier studies have shown, the F3 pheromone can be delivered to cats in a variety of ways to promote adaptation, but some devices may have limitations in use. For example, plugged diffusers require electricity to effectively deliver the pheromonal message and should be plugged in for approximately 24 h to have an impact on animals. Sprays or mists act more quickly and can be applied to specific locations; however, when alcohol-based, sprays need to be applied sufficiently in advance to allow the alcohol to evaporate. Working with a water-based mist can address these limitations, as it can be applied in the animal’s presence without harming it, which can be useful; for example, during car rides. Moreover, compared with alcohol-based mists, water-based mists deliver pheromones more efficiently, as the water in the microdroplets evaporates almost immediately, releasing the pheromone they contain and making it detectable. Hence, the aim of this study was to test the effect of a water-based mist form of a synthetic analogue of the F3 facial pheromone on the behaviour of cats facing a challenging situation: introduction to a new environment and the presence of an unknown human. Based on previous studies investigating the effects of F3 application, we hypothesise that when the room is treated with F3, cats will be more willing to interact with the unknown human and display more exploratory behaviours.

Materials and methods

The study protocol was approved by the institutional ethics committee of the Research Institute (approval number CE_2022_08_F3b_02).

Animals

The study was carried out on 22 adult domestic cats aged 4–15 years. This population contained 10 males (three intact and seven castrated) and 12 females (six intact and six spayed) from the research institute’s cat housing facility. The 22 cats were divided into two groups of 11, balanced for their age, sex, neuter status and personality. The personality of each animal was assessed by an animal caretaker using a visual analogue scale (VAS) to score their fearfulness and sociality towards humans as well as their adaptability to a new situation. VASs are used to assess the personality of various animal species and provide a practical and sensitive tool for behavioural assessment.^20
–22 Nevertheless, its use involves a degree of subjectivity due to the observer’s personal interpretation of cats’ behaviours. For this study, all VASs were completed by the same caretaker who worked daily with all the cats.

For this study, only healthy animals who were able to express all behavioural patterns of the species were included. The animals’ health and welfare were guaranteed by close monitoring by animal keepers and veterinarians, thanks to a monthly Animal Welfare Assessment performed by the Animal Welfare Body of the Research Institute.²³

Experimental design

The aim of this study was to investigate the behaviour of cats exposed to an unknown human in a new environment, while the experimental room had been previously treated or not with a mist-form analogue of the F3 FFP. To avoid any contamination between groups, two identical rooms were used to test the cats’ behaviours, with the same treatment always applied in the same room. Hence, the procedure took place in a 7.4 m² test room fitted with ventilation, in which two toys, a water bowl, a food bowl and a litter box were placed (Figure 1). To replicate a real living room, the experimental room was furnished with a hiding place, a plastic stool, a coffee table and a shelf.

Figure 1

Schematic illustration of the experimental room. The water-based mist treatment, placebo or F3, was applied 10 mins before the cat entered the room at the four points marked by the dotted circles. After entering, the unknown human turned on the timer and sat down, cross-legged, inside the human contact area marked out by black tape on the floor

The treatment used during this study was a mist form of an analogue of the F3 FFP, which is composed of a mixture of oleic, azelaic, pimelic and palmitic acids.¹² The product is a water-based mist, an emulsion of 2% (volume per volume) F3 pheromone, while the control treatment consisted of the same excipients but without the active compound. This formulation is patent pending (US-2025-0312248-A1) and does not require any shaking.²⁴ The treatment was applied 10 mins before the cat entered the room. Cats were all tested individually: half of them (F3, n = 11) were tested after application of the F3 mist, and the other half (controls, n = 11) were tested after application of the placebo. Treatments were sprayed on the plastic stool, shelf, coffee table and hiding box (Figure 1), with each treatment point sprayed four times. The procedure was double-blinded and randomised.

As introduction to a new place can be a stressful event for cats, exposure to the unknown human was delayed by 40 mins after the cat entered the room. This was done to discriminate between behaviours related to exposure to a new environment and behaviours linked to the presence of an unknown human. Hence, each cat was brought into the experimental room in a transport cage, the operator opened the cage, let the cat out and then left the room. Once the cat was alone in the room, a timer was started and, after 40 mins, a human (woman aged approximately 25 years) who had never had contact with the tested cats entered the room, calmly walked directly to the timer on the wall of the experimental room to start it, and then sat down, cross-legged, inside the human contact area. The human was the same person throughout the experiment and remained in the room for 10 mins: during the first 5 mins, the human remained passive; then, the human extended her arm, palm downwards, towards the cat while calling its name. If the cat approached, the human briefly scratched its head. This contact attempt was repeated after 2 mins 30 s. The contact attempt was not performed if the cat was already in physical contact with the human.

Between cats, the experimental rooms were cleaned using a standardised protocol taking around 15 mins. This protocol included cleaning the floor and the different objects placed in the room using a degreasing detergent (Vigor, diluted at 30 ml/l).

Behavioural analysis

Cats’ behaviours were analysed for 10 mins, starting when the unknown human sat down in the human contact area. All tests were filmed using a GoPro Hero 9 camera fixed to the wall of the experimental room. Videos were then analysed by two independent readers trained in animal behaviour observation using Behavioral Observation Research Interactive Software (BORIS).²⁵ Behaviours were analysed as frequency (f), duration (d) or latency (l). For latencies, if a cat never reached the criterion, the maximum time duration (ie, 600 s) was applied (Table 1).

Table 1

Behaviours observed during the video analysis²⁵

Category	Behaviour	Description
Towards human	In the human contact area (f, d, l)	Any part of the cat’s body crosses the line drawn on the ground representing the zone of contact with the unknown human
Towards human	Physical contact with the unknown human (l)	The cat voluntarily touches the unknown human with any part of its body
Towards environment	Rubbing (f)	The cat rubs any part of its body against an object
Towards environment	Feeding (f)	The cat eats the dry food from the bowl
Locomotion	Move (f, d)	The cat moves, whatever their gait or activity
	Sit (f, d)	The cat sits upright with its hindlimbs bent and its forelimbs straight
	Stay (f, d)	The cat does not move, four paws are on the ground, limbs extended or bent, with the belly either touching the ground or not

d = duration; f = frequency; l = latency

Statistical analysis

Data analysis was performed using R version 4.2.2 and RStudio version 1.4.1103.²⁶ Statistical significance was set at P <0.05. Treatment-related groups were homogeneous for age, sex, neuter status, scores related to VASs (adaptability, confidence, sociability) and participation in previous studies. For age, adaptability, confidence and sociability, comparisons between the two groups were assessed using Student’s t-test if conditions of data normality (graphically and using normality tests) and homogeneity of variances (using Fisher’s test) were verified. Otherwise, a Wilcoxon test was preferred. For sex, neuter status and their possible participation in a previous study, Fisher’s exact tests were applied.

Before starting the statistical analysis of the behavioural parameters, inter-observer reliability was checked using Pearson’s or Spearman’s correlation coefficient, depending on whether the normality assumption was met. Correlations had to exceed 80% for a behaviour to be analysed.

The effect of treatment was evaluated using mixed models, with the date of the cat’s passage included as a random effect. For some behaviours – ‘Sit’ (d), ‘Stay’ (d) and ‘Rubbing’ (f) – an effect of the housing room was detected and was therefore also included as a random effect. An effect of previous participation in a study was detected for some behaviours, so this parameter was also included as a random effect. For behaviours expressed as durations, general linear mixed models (GLMMs) were performed. Normality and homoscedasticity of model residues were assessed using graphical representation and normality tests. When these assumptions were not met on raw data, data were transformed using a Box-Cox transformation to satisfy these conditions. For behaviours expressed as frequencies, generalised linear mixed models (GsLMMs) were performed, with the Poisson distribution as first intention. Data dispersion was verified using the χ²/degree of freedom [DF] indicator. When this indicator exceeded 2, indicating overdispersion, the negative binomial distribution was used instead. A frailty model for survival data was applied to behaviours expressed as latencies.

In addition to these analyses, a multivariate approach was applied using principal component analysis (PCA). Only data with a probability (P) value less than 0.20 from the univariate analysis were included. The qualitative treatment variable was incorporated as a supplementary variable to assess its effect on data variability. A biplot was used to visualise the relationships between individuals and variables, while the contribution, correlation and cos² of each variable were examined for each selected principal component to provide a deeper understanding of their role in explaining variability in the dataset.

Results

Univariate analysis

Behaviours towards environment

Cats in the F3-treated situation exhibited significantly more rubbing against objects (GsLMM, χ² = 14.056, DF = 1; P <0.001) than those in the control group (Figure 2). No effect of the treatment was observed on feeding behaviour (GsLMM, χ² = 1.9014, DF = 1; P = 0.168).

Figure 2

Graphical analysis of occurrences of cats’ exploratory behaviours (sit, move, stay and rubbing) excluding interactions with humans. The water-based mist treatment, control (white, n = 11) or F3 (grey, n = 11), was applied 50 mins before the unknown human entered the room. Data are presented as mean ± SD. *P <0.05, ***P <0.001). NS = non-significant

Locomotion

Cats in the F3-treated situation sat more frequently than those in the control group (GsLMM, χ² = 6.058, DF = 1; P <0.05) (Figure 2). Similarly, F3-treated cats moved more regularly than those in the control group (GsLMM, χ² = 6.163, DF = 1; P <0.05) (Figure 2). For both behaviours, total duration did not differ significantly between the two groups of cats (Table 2). Regarding the ‘stay’ behaviour, neither frequency (GsLMM, χ² = 0.259, DF = 1; P = 0.611) (Figure 2) nor total duration (Table 2) differed significantly between the two groups.

Table 2

Total duration of the sit, move and stay behaviours did not differ between groups

Behaviours	Control group (n = 11)	F3 group (n = 11)	Statistical results
Sit	85.52 ± 98.55	110.11 ± 103.67	GLMM, χ² = 0.765, DF = 1; P = 0.382
Move	157.93 ± 134.34	201.52 ± 79.66	GLMM, χ² = 2.558, DF = 1; P = 0.11
Stay	210.44 ± 201.41	146.84 ± 114.38	GLMM, χ² = 0.833, DF = 1; P = 0.361

Data are mean ± SD unless otherwise indicated

DF = degrees of freedom; GLMM = general linear mixed model

Behaviours towards a human

No statistically significant differences were found between the two groups for the behaviours towards the human. Trends were observed for cats in the F3-treated situation to be quicker than control cats to enter the human contact area (frailty model, χ² = 3.54, DF = 1, P = 0.06) as well as to initiate the first physical contact with the unknown human (frailty model, χ² = 3.454, DF = 1, P = 0.063) (Figure 3). The number of entries in the human contact area (GsLMM, χ² = 0.593, DF = 1, P = 0.441) and the total time spent in it (GLMM, χ² = 0.016, DF = 1, P = 0.90) did not differ between the control and F3-treated cats (Figure 3).

Figure 3

Graphical analysis of the cats’ average behaviour towards the unknown human. The water-based mist treatment, control (white, n = 11) or F3 (grey, n = 11), was applied 50 minutes before the unknown human entered the room. Data are presented as mean ± standard deviation. (t: P <0.07)

Principal component analysis

A PCA was conducted as an exploratory approach; hence, it was decided to only include in the analysis variables presenting a P value less than 0.20 in the univariate analysis as stated in Table 3.

Table 3

Statistical P values of the parameters included in the principal component analysis

Behaviours	Control group (n = 11)	F3 group (n = 11)	P value
Human contact area (l)	260.09 ± 290.49	81.17 ± 164.80	0.060
Human first contact (l)	275.13 ± 281.49	93.51 ± 120.04	0.063
Sit (f)	3.36 ± 3.92	5.18 ± 2.70	0.022
Movement (f)	13.45 ± 8.30	16.55 ± 8.24	0.013
Movement (d)	157.93 ± 134.34	201.52 ± 79.66	0.110
Feeding (f)	0.14 ± 0.32	0.41 ± 0.97	0.168
Rubbing (f)	2.23 ± 2.25	6.55 ± 8.23	0.0002

Data are mean ± SD unless otherwise indicated

d = duration; f = frequency; l = latency

The correlation, contribution and cos² of each variable for the two principal components of the PCA are shown in Table 4 and are graphically represented in Figure 4. A total of seven components were extracted from the analysis, but only the first two were interpreted as they explained 74.53% of the total variation (Table 4). The first component (PC1) explained 52.99% of the behavioural variation, while the second component (PC2) accounted for 21.54% of the behavioural variation. PC1 was mainly correlated with locomotion (sit, move) and human interactions (latency to enter the human contact area and latency to touch the human). We observed that the cats who were quicker to interact with the unknown human were those exhibiting more locomotor activity. Analysis of PC1 also revealed that control cats were more heterogeneously distributed across this axis than those treated with the F3 water-based mist. PC2 correlated with feeding and rubbing behaviours, showing that the cats eating more frequently were also the ones rubbing more often.

Table 4

Parameters for the first two principal components (PCs) (PC1 and PC2)

Behaviours	PC1 (52.99%)			PC2 (21.54%)
Behaviours	Correlation	Contribution (%)	Cos²	Correlation	Contribution (%)	Cos²
Human contact area (l)	–0.847	19.33	0.717	0.338	7.56	0.114
Human first contact (l)	–0.830	18.58	0.689	0.340	7.70	0.116
Sit (f)	0.766	15.80	0.586	0.066	0.29	0.004
Movement (f)	0.874	20.59	0.764	–0.056	0.21	0.003
Movement (d)	0.846	19.27	0.715	0.020	0.03	0.0004
Feeding (f)	0.331	2.96	0.110	0.786	41.02	0.618
Rubbing (f)	0.359	3.47	0.129	0.807	43.20	0.651

The most relevant traits for each PC are shown in bold

d = duration; f = frequency; l = latency

Figure 4

Biplot for the two principal components of the principal component analysis (PCA). Control cats (n = 11) are represented by red circles and F3-treated cats (n = 11) by green triangles. Arrows represent the correlation between each variable and the axis. d = duration; f = frequency; l = latency

Discussion

Our results suggest that using a water-based mist form of a synthetic analogue of the facial F3 pheromone can improve cats’ coping abilities while facing challenging situations. During our study, cats were individually exposed to an unknown human entering the room after 40 mins. The main finding is that cats receiving the pheromone exhibited more rubbing on objects than those receiving the placebo. By performing this behaviour, cats deposit their own facial pheromones into their environment – a behaviour typically associated with feeling comfortable in their surroundings.²⁷ We can therefore assume that the pheromone treatment helped the cats feel more at ease in this unfamiliar environment and less disturbed by the presence of a human. Although none of the behaviours directed towards the human reached statistical significance and our sample size was limited (n = 22), observed trends towards shorter latencies for cats in the F3-treated room to enter the human area and initiate contact are consistent with the idea that these cats were more at ease in this novel situation.

In a previous study investigating cats’ temperament, the authors observed that the friendliest cats tended to have a calm/active profile.²⁸ This is consistent with our analysis, as F3-treated cats tended, as noted earlier, to exhibit more friendly behaviour by being more willing to interact with the human while also being more active. Although the total time spent sitting, moving or standing did not differ between groups, cats in the F3-treated situation were more likely to switch between activities and engage more frequently in both ‘move’ and ‘sit’ behaviours. The duration of sitting may be influenced by various factors, such as boredom, resting, observation or grooming, depending on context.²⁹ In contrast, the frequency of sitting can reflect transitions from movement to a resting state. An increased frequency of movement may indicate more relaxed transitions between resting and active states, suggesting comfort within the environment and a greater willingness to explore. The increased number of transitions between ‘sit’ and ‘move’ behaviours in F3-treated cats may reflect increased exploratory behaviour, consistent with the observed increase in rubbing behaviours. Cats who move and sit more frequently are changing activity more often. We can hypothesise that those cats explore their environment more frequently, potentially visiting different spots in the room and triggering rubbing behaviour. A precise analysis of spatial movement would have been valuable to confirm this hypothesis. The PCA was performed as an exploratory tool to visualise patterns of behaviours and should be interpreted cautiously. It suggested that cats more likely to change posture were also quicker and more willing to engage with the unknown human, indicating that changing posture may be linked to exploration of the physical environment, including the unknown human. Given our limited sample size and the lack of statistical significance for behaviours directed towards the human, these results need confirmation in a more robust study. Stress is known to reduce cats’ exploratory activities and interaction with humans.³⁰ Our observation of increased exploration and a trend towards more human interactions in F3-exposed cats further supports the pheromonal mist’s potential to promote coping and reduce behavioural signs of stress. This is also consistent with our observations on feeding and rubbing behaviours, two parameters that are negatively impacted under stress.³⁰ In our study, cats who rubbed more also ate more and were more likely to have received the F3 treatment. Therefore, F3-exposed cats appeared more comfortable during the test, likely reflecting improved coping strategies and faster adaptation to this new situation.

Taken together, these observations indicate that cats treated with the F3 water-based mist were more curious, confident and potentially more sociable towards humans, consistent with findings from other studies on the F3 pheromone. The F3 pheromone in spray form is known to facilitate cats’ adaptation to unfamiliar surroundings.^19,31 Cats receiving an F3 analogue treatment were less likely to vocalise during the acclimation period before a veterinary examination compared with placebo-treated cats.³² The stress-reducing effects of an F3 analogue have also been confirmed in a recent study of cats during transportation.¹³ These behavioural findings align with studies monitoring cortisol levels, which showed a reduction in F3-treated cats.¹⁹ Therefore, our F3 treatment demonstrates similar efficacy to other F3 pheromonal products while offering a distinct profile that may be useful in specific situations. The mist form allows for targeted application with rapid effect compared with passive diffusers. In addition, its water-based formula makes it safe for use in the presence of animals, whereas alcohol-based sprays require a waiting period for the alcohol to evaporate before the animal can be introduced.

One limitation of our study is the number of cats involved (n = 22), which may have limited our ability to detect a significant effect of F3 on behaviours directed towards the unknown human. Although these effects did not reach statistical significance, trends suggested shorter latencies for entering the human area and for first physical contact with the unknown human in F3-treated cats (P <0.07). Future studies with larger sample sizes, ideally including owned cats living in home environments, are needed to strengthen the robustness of our observations and to confirm and extend our results.

Regarding the PCA, F3-treated cats appeared more homogeneous in their interactions with humans, whereas control cats showed greater variability, with some approaching and touching the unknown human more slowly or quickly. However, this observation should be interpreted with caution given our limited sample size (n = 22) and the lack of statistically significant effects on human-directed behaviours. It is possible that this variability reflects individual differences in sociability. Semiochemicals may therefore ‘smooth out’ these personality differences, making cats more sociable even if this is not their inherent temperament, which could facilitate the adaptation process.

Conclusions

Overall, the presence of the F3 water-based mist appeared to have a positive effect on cats’ behaviour, with the most robust outcome being increased rubbing in cats in the F3-treated room. Although effects on human-directed behaviours did not reach statistical significance – likely due to the small sample size – observed trends and other results suggest that the treatment may help cats to cope with challenging situations by encouraging their natural behaviours. The pheromonal treatment may promote confidence, making cats more willing to interact with an unfamiliar human. This raises the potential for using the product in situations where cats encounter unfamiliar humans, such as during adoption, when introducing a new family member, hosting guests or engaging a pet sitter. Moreover, because this F3 mist is water-based, it is safe for both animals and owners.

Footnotes

Acknowledgements

We would like to thank Syria Cellai for her participation in the study and for serving as the unfamiliar human during all experiments, and Umut Burak Agan for his help with manuscript preparation. We also thank all members of the Vertebrate Animal Study Service for their support, care of the cats and contribution to the study, as well as SIGNS Labs for providing the products free of charge. In addition, we thank Dr Rajesh Durairaj for his help in improving the manuscript.

Accepted: 7 January 2026

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Ethical approval

The work described in this manuscript involved the use of experimental animals and the study therefore had prior ethical approval from an established (or ad hoc) committee as stated in the manuscript.

Informed consent

Informed consent (verbal or written) was obtained from the owner or legal custodian of all animal(s) described in this work (experimental or non-experimental animals, including cadavers, tissues and samples) for all procedure(s) undertaken (prospective or retrospective studies). No animals or people are identifiable within this publication, and therefore additional informed consent for publication was not required.

ORCID iD

Manon Chasles

Alessandro Cozzi

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