Salivary Cortisol Levels and Stress in Adults with Profound and Multiple Intellectual Disabilities Participating in Structured Aquatic Dance Intervention: A Randomized Controlled Crossover Trial


This study is part of the SWAN research project, a multicenter randomized intervention study, including participants from different habilitation centers in four Swedish regions. Further details of the study design were previously reported in a study protocol.24. In each center, there were two groups: an early intervention group (group 1) and a late intervention group (group 2). Participants were randomized to group 1 or 2 by minimization27 using MINIM software28. Group 1 received SWAN when entering the study; Group 2 served as a control group. After a two-month washout period, group 2 received the same intervention as group 1, while group 1 resumed normal activities. Therefore, all participants met the intervention and control conditions. For a CONSORT flowchart, see Fig. 1.

Figure 1


Participants were recruited from habilitation centers in four regions of central Sweden by physiotherapists in collaboration with the research team, according to the inclusion criteria: being an adult (18 to 65 years old) with PIMD and having previous experience of hydrotherapy or other aquatic activities without any discomfort. PIMD was defined as having a motor impairment corresponding to level IV or V of the gross motor function classification system29 and profound intellectual disability according to the International Classification of Diseases, Tenth Revision of the World Health Organization30. The definition was communicated to local physiotherapists and, where necessary, medical records were checked for clarification. Exclusion criteria were: severe hearing impairment or deafness (since music was played to enhance water dance activity) and infections or ulcers, as this could be infectious in the pool. Cerebral palsy and epilepsy were the most common diagnoses among participants. These diagnoses were often present simultaneously and for some participants in combination with other diagnoses, such as autism spectrum disorders, other brain disorders, rare diseases, asthma/allergy or visual impairments.

A pre-study power analysis using an online service ( reported that 40 participants were needed to achieve 80% study power at 5% alpha. However, it was only possible to recruit thirty-six people. Two of them withdrew their participation at the beginning of the study; one withdrew before the first session and the other after the first SWAN session due to stomach issues and group activity tolerance issues. Thus, 34 people completed the intervention, including 20 men and 14 women. The characteristics of the study participants are shown in Table 1.

Table 1 Characteristics of study participants.

Structured Aquatic Dance Intervention

The SWAN intervention is a group aquatic activity with four to five participants in a heated swimming pool (33-35°C). The 40-minute sessions took place around noon once a week for 12 weeks. Two of the four SWAN groups were scheduled in the morning (around 11am) and the other two groups were scheduled in the afternoon (around 1pm). Each session followed a structured program focusing on the following key elements: experience of dance and music, adapted movements, stimulation of the senses and social interaction. A playlist of nine pieces of music has been created to stimulate the rhythm of the movements performed, promote relaxation and arouse different emotions. Two assistants accompanied each participant; one of them was in the pool and served as a dance partner while the other remained poolside to support when participants entered or exited the pool. The second assistant also helped with floating devices and other practical matters. The sessions were led by two instructors. One of them instructed the group from the edge of the pool and the other was in the water to guide the participants and assistants with the dance moves.

Outcome measures

Saliva sample

Twenty-six samples were to be collected for each participant, 16 samples during the intervention condition and 10 samples during the control condition. The schedule for collecting saliva samples is shown in Table 2.

Table 2 Schedule for collection of saliva samples.

Saliva was collected morning and evening at the participants’ homes by caregivers and at the swimming pool as part of the intervention of a physiotherapist from the research group. At all sites, salivary cortisol was collected using collection equipment (Salimetrics LLC, State College PA) comprising a swab (SalviaBio Children’s Swab) and a centrifuge tube.

For salivary cortisol sampling at participants’ homes, sampling kits were provided and completed with disposable gloves for hygiene reasons. All tubes were pre-labeled with a number specific to each participant and a number representing the occasion on which the sample was to be collected. Sampling kits were distributed to participants’ homes with written step-by-step instruction and a diary to record sampling information (date and time) and contact information for questions. Further instructions were given if needed with a short informational video clip from Salimetrics on how to perform the saliva collection. In addition, an informational video on how to register/handle samples was distributed and made available to all caregivers or assistants of participants. Collection of saliva samples from adults with developmental disabilities with the assistance of a parent or caregiver has been found to be feasible17. Home saliva samples were collected equally for all participants, whether they belonged to the intervention or control condition. The samples were taken in the morning and evening of the day, one week before the first session; the days of the 1st, 6th and 12th sessions; and the same day, a week after the last session. Morning saliva was collected in bed after waking up and before participants brushed their teeth or ate breakfast, typically between 7:00 a.m. and 8:30 a.m. (Md = 8:00 a.m.). Evening saliva was collected at least one hour after dinner or another meal and before brushing teeth, usually between 8:00 p.m. and 9:00 p.m. (Md = 8:17 p.m.). Saliva samples were immediately frozen at participants’ homes (−18°C) and later, as part of post-intervention measures, transported in a freezer bag (0°C) between one and three hours to the laboratory where they were stored in an ultra-low temperature freezer (−80°C).

Sampling equipment and a sampling procedure similar to that used in participants’ homes was used at the swimming pool in the intervention, i.e. the swab was placed in the mouth of the participant for 60–90 s, then placed in the centrifuge tube and labeled with the participant ID, date, and time. Intervention samples were collected during sessions 1, 6, and 12, just before entering (baseline) and leaving the pool, respectively. The saliva collected at the end therefore reflects the first part of the SWAN sessions. Samples taken from the pool before and after the SWAN sessions were placed in a freezer bag (0°C) and between one and four hours later placed in an ultra-low temperature freezer (−80°C) at laboratory.

Salivary cortisol analysis

All saliva samples were tested for cortisol in duplicate using a highly sensitive enzyme immunoassay (Salimetrics, State College, PA). The test used 25 µL of saliva per determination. The lower limit of sensitivity was 0.2 nmol/L. The mean intra-assay coefficient of variation was 5% and the mean inter-assay coefficient of variation was 7%. A random selection of 9% of the samples was analyzed twice to determine the reliability of the test. The correlation was r = 0.95 (p

Assistant Ratings

The two assistants who accompanied the participants rated the degree of stress of the participants in agreement on a 5-point Likert scale, from 0 = no stress at all, to 4 = a lot. We haven’t explained any particular signs to look for except for signs of stress in general. Instead, we asked the assistants, who all knew the participant well, to be alert to the participant’s specific signs (sound, facial expressions, and motor behavior) indicating stress. The evaluation was carried out three times in each session: before the start of the session when the participant was in the locker room, during the water dance session when the participant was in the swimming pool and finally after the session when the participant was back in the changing room.

statistical analyzes

All analyzes were performed using the IBM Statistical Package for the Social Sciences for Windows, version 25.0 (IBM Corporation, Armonk, NY, USA). Raw cortisol values ​​were non-normal according to the Shapiro-Wilk test (p 31. Thus, all the analyzes were performed on ln-transformed data; however, untransformed data has been used in figures and tables for clarity. The ln-transformed values ​​were approximately normally distributed with an skewness of −0.006 with a standard error of 0.088 and a kurtosis of −0.394 with a standard error of 0.176. No outliers were identified. Linear Mixed Model (LMM) analysis with restricted maximum likelihood was chosen because it uses all the information available in the data in a repeated measures design and is robust in dealing with missing data32. A separate linear mixed model was constructed for the two outcome variables, assistant ratings and salivary cortisol. All models included random intercepts and unstructured component analysis was used to account for within-subject correlation over time. Significance thresholds were set at 0.05 (two-sided).

Ethical considerations

The study was carried out in accordance with the Declaration of Helsinki. Regarding the profound disability of the participants, all had a legal guardian who gave written informed consent after reading information about the study and receiving information from the researchers by phone or in person. The risk of discomfort or harm to participants was not considered to be higher than with regular treatment. Nevertheless, if observed, participants’ assistants and legal guardians should be alert to signals of discomfort or harm and immediately notify the SWAN manager or researcher in charge. No harm or discomfort was reported except for the discomfort of the person pulling out after the first session. The study was approved by the Regional Ethics Review Board in Uppsala, Sweden (dnr: 2018/070).

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