Materials and reagents

Non-effervescent Haz-Tab (4.5 g NADDC per tablet) (Guest Medical, catalog number: H8801) made up to 1,000 ppm (one tablet per 2.5 L)
The Positive and Negative Affect Schedule (PANAS) (Watson et al., 1988)
Appropriately sized hospital scrubs for each participant
Blankets

Equipment

MRI machine (3-Tesla Siemens Magnetom Lumina, 32-channels head-coil)
Water heater (Laz-y-spa, UK)
Water pump (Laz-y-spa, UK)
Water chiller unit (Grant Instruments, UK)
Three-lead electrocardiogram (ECG, Fukuda Denshi, UK)
ECG electrodes (Ambu^® Blue Sensor P, Malaysia)
Electrodes covered with Tegaderm dressings (3M, Germany)
35 mm respiratory hose (Cranlea, UK)
Reusable nose clip (Cranlea, UK)
Two-way T-shape non-rebreathing valve (Hans Rudolf, US)
Respiratory turbine (K.L. Engineering, US)
Thermistor pod (ML309, AD Instruments, Australia)
Skin temperature thermistor (MLT422/A, Australia)
Digital recorder (Powerlab, AD Instruments, Australia)
Laptop connected to the Powerlab and programmed to a sampling rate of 400 Hz (Chart software, AD Instruments Australia)

Scanning parameters

Anatomical scan: T1-weighted MPRAGE images with the following parameters: repetition time (TR) = 1,900 ms, echo time (TE) = 2.74 ms, flip angle = 8°, field of view (FOV) = 256 × 256 mm², voxel size = 1.0 × 1.0 × 1.0 mm³, and 192 axial slices.
Functional scan: blood oxygenation level–dependent (BOLD) contrast whole-brain functional images were acquired using a T2-weighted gradient-echo Echo Planar Imaging (EPI) sequence with a 32-channel head coil. Sequence duration 13.12 min. Acquisition parameters: TR = 2,680 ms; TE = 30 ms; matrix size = 64 × 64 mm²; slice thickness = 2.5 mm, spacing between slices = 2.5 mm, flip angle = 80°; 292 volumes with 48 axial slices were measured in interleaved slice order and positioned along a line to the anterior-posterior commissure (AC-PC orientation). An automated high-order shimming technique was used to maximise magnetic field homogeneity.

Software

MATLAB 2022a (MATLAB and Statistics Toolbox Release 2012b, The MathWorks, Natick, Massachusetts, United States)
CONN (v.2021a): A functional connectivity toolbox for correlated and anticorrelated brain networks (Whitfield-Gabrieli and Nieto-Castanon, 2012, http://www.nitrc.org/projects/conn)
JASP (JASP Team, 2022, version 0.16.3) (Computer software)

Procedure

Step 1. Participant screening
1. Eligibility screening
  1. Provide interested volunteers with a participant information sheet containing sections explaining eligibility criteria, procedures, and risks.
  2. Eligibility criteria: (i) age is between 18 and 45 years old; (ii) no history of medical, neurological, psychiatric, or substance use disorders; (iii) naïve to CWI (not exposed to cold water including cold shower, unheated swimming pool, or the sea) in the last 12–18 months, and (iv) free from chronic pain of any type (Figure 1).
    
    Figure 1. Participants selection consort diagram. The diagram provides information about the selection steps (on the left) and the number of participants excluded in each selection step.
  3. If required, screen participants by telephone interview and undertake any additional pre-screening tests required by the medical officer to rule out any significant history of medical, neurological, psychiatric, or substance use disorders.
2. Claustrophobia screening
  1. Siemens Lumina scanner has a relatively large tunnel, is fully lit, and is open at both ends. This dramatically lowers the experience of claustrophobia. Ask participants whether they have ever experienced claustrophobia (or fear of being in a closed space) in the past.
  2. Ask participants whether they have previous experience with MRI scans.
  3. Ask participants whether exposure to a feared situation have led to immediate anxiety or panic attacks in the past.
  4. If the answer is yes to a. or c., the participant does not pass claustrophobia screening.
3. MRI compatibility screening
  1. MRI compatibility screening should comply with the standard operation procedures approved for scanner use. The procedures can be slightly different across universities. Discuss the screening procedures with your local scanner manager.
  2. Ask participants to answer questions assessing their eligibility for fMRI study: (i) Do you have a heart pacemaker or pacing wires? (ii) Have you had any recent surgery? (iii) Have you had any surgery to your head (including eyes/ears/brain)? (iv) Do you have any implanted devices (e.g., programmable hydrocephalus shunt, nerve stimulator, cochlea implant, aneurysm clip? (v) Have you ever sustained any injuries involving metal to your eyes or any parts of your body? (vi) Do you have any of the following: dentures with metal, body piercings, hearing aid, nitro patch, artificial limb or prothesis, tattoos? (vii) For women: do you have an IUD (coil)? Could you be pregnant?
  3. If the answer to all questions is no, the participant will be taken to the next screening step. If the answer to one or several questions is yes, take this case to a radiographer.
Participants who passed Step 1 were scheduled to visit the Institute of Medical Imaging and Visualisation (IMIV) at Bournemouth University where the study took place. For the scheduled visit, ask participants to (i) refrain from eating food 2 h prior to the scanning session; (ii) bring a swimsuit/trunks or shorts (+vest top for women), a towel, a woollen jumper or sweatshirt with no metal, and slippers or flip flops.

Step 2. Pre-CWI assessments
1. MRI safety assessment
  1. If the participant passes the MRI safety assessment, explain the experimental procedures and familiarise participants with the setup, including cold-water tub and monitoring equipment.
  2. Answer any questions the participant may have and ask them to complete a written informed consent form.
2. Pre-CWI mood assessment
  1. Provide a printed version of the PANAS questionnaire and instruct them to evaluate all items of the questionnaire, reflecting on how they feel as they complete it (in the moment).
  2. Ask participants if they have any questions. Ensure that their clothes do not have any metal parts (or provide a set of scrubs and blankets).

Step 3. Experimental procedures
1. Pre-CWI scan
  1. Position a participant in the scanner (Figure 2). Use MRI-compatible headphones to reduce scanner noise. Use pads to fixate the participant’s head to reduce head movements.
    
    Figure 2. Participant’s position in the scanner
  2. Provide an alarm button (by pressing the button, participants are able to stop the scanning at any time).
  3. Ask participants to keep awake and keep their eyes open.
  4. Start anatomical (structural) scan (4.5 min). Immediately after, start the functional scan (resting state) (13.12 min).
  5. After completing the pre-CWI scanning procedures, take the participant to a changing room to change into swimwear.
2. CWI (cold-water immersion procedure)
  1. Record participant’s height and mass.
  2. Instrument with a three-lead ECG connected via ECG electrodes and waterproof the electrodes using Tegaderm dressings to improve the signal quality during immersion.
  3. Provide the participants with a nose clip and mouthpiece and ask them to breathe freely throughout.
  4. A minimum of 2 min of baseline data are collected on the Chart software with the participant seated next to the cold-water tank. The Chart program also collects water temperature, ECG, and ventilatory data throughout the immersion, and comments should be added to the chart software to highlight pre immersion, immersion, and post immersion phases.
  5. Participant will stand, immediately enter the tank, and be seated in the water within a 10–15 s period. They will immerse to the depth of the axilla (see image in Graphical overview). If the immersion is too deep, ask them to sit at the shallow part; if it is too shallow, ask them to sit in a deeper part. This is done by putting weights on the bottom, which the participants could sit on.
  6. Participants are encouraged to breathe freely through the mouthpiece and do not attempt to hold their breath.
  7. Monitor the ECG during immersion for any ECG abnormalities that would necessitate withdrawal from the cold water.
  8. Upon the completion of 5 min of immersion or if the participant requires earlier withdrawal, they stand up, exit the water tank onto a skid resistance surface, and the ECG and mouthpiece are removed.
  9. Participants remove their swimwear and dry off and dress in private, as quickly as possible.
  10. Record the duration of time from exiting the cold-water tub to the MRI chamber door closing, following the positioning of the participant in the scanner. The average time between exiting the cold-water tub and entering the scanner was M = 5.82 min, SD = 2.08 min (range: 2 min 25 s–10 min 30 s). It is not anticipated that the transition time between the tub and the scanner dramatically affects changes in the brain, as previous research reported the effects of CWI to last over a few days (Massey et al., 2022). However, we encouraged participants to dry off and dress quickly after the CWI. It must be noted that the cold-water tub should be set up as close to the MRI scanner as possible, to ensure the shortest transition time. In our study, the cold-water tub was next door to the scanner room.
3. Post-CWI scan
  1. Position the participant in the scanner (Figure 2).
  2. Use a lightweight cotton blanket to cover the participant’s body (to minimise shivering after CWI).
  3. Switch off the built-in scanner ventilator (to minimise participant cooling and body movement due to cold air).
  4. Immediately after, start the functional scan (resting state) (13.12 min).
Table 1 summarises the timeline for the experimental steps to provide a detailed estimation of the complete study per participant at the MRU suite.

Table 1. Detailed timeline for experimental steps B and C at the MRI suite

Steps	Sub steps	Additional time between sub steps	Time
B	MRI safety assessment (including explanation of CWI procedure and signing consent form)		5 min
	Pre-CWI mood assessment		2–3 min
		Changing into MRI-compatible clothes (if required)	1–3 min
C	Position participant in the scanner (pre-CWI)		3 min
	Pre-CWI scanning		4.5 min + 13.12 min = 17.62 min
		Localise scan to position the brain	1 min
		Get participant from the scanner, change into swimwear	3–4 min
		Instrument with ECG electrodes, nose clip, and mouthpiece	2–3 min
		Taking baseline measurements	2–3 min
	Cold-water immersion		5 min
		Remove ECG, nose clip, and mouthpiece	1 min
		Drying and changing into MRI-compatible clothes	M = 5.83, SD = 2.09*
	Position participant in the scanner (post-CWI)		2–3 min
	Post-CWI scanning		13.12 min
		Localise scan to position the brain	1 min
	Total time:		~62 min**

*This parameter ranged between 2 min 25 s and 11 min 15 s.

**Total time can be reduced if participants arrive in MRI-suitable clothes.

Data analysis

The original paper provides detailed data analysis for this study. The analytical steps are summarised in Figure 3.

Figure 3. Analytical steps in the present study

Tips

To disinfect the mouthpieces, nose clips, and respiratory hose, two non-effervescent Haz-Tab were dissolved into 5 L of cold tap water. The tablet was allowed to dissolve completely before the equipment was dismantled and submerged in the liquid for 20 min.

The CWI tub does not need to be drained or disinfected between participants. It was checked twice a day for pH and free and combined chlorine and was dosed with chlorine daily in accordance with the manufacturer’s guidelines. The tub also had filters for trapping hair and detritus, which were cleaned daily.

Validation of protocol

Yankouskaya et al. (2023). Short-term head-out whole-body cold-water immersion facilitates positive affect and increases interaction between large-scale brain networks. Biology, 12(2): 211.

Acknowledgments

We wish to thank the participants and radiography team who are not authors (Carlo Vitale and Theophilus Akudjedu). This work was supported by the Institute of Medical Imaging and Visualisation (Bournemouth University, UK).

Competing interests

None of the authors has any conflicts of interest.

Ethics

The protocol requires approval from a local ethics committee. The study was reviewed and approved by the Departmental Research Board and Ethics Committee at Bournemouth University (Ethics ID 34976 17.02.2021) and conducted in accordance with the Declaration of Helsinki. Written informed consent from each participant was obtained before the study.

References

Bitar, N., Dugré, J. R., Marchand, S. and Potvin, S. (2020). Medial Orbitofrontal De-Activation During Tonic Cold Pain Stimulation: A fMRI Study Examining the Opponent-Process Theory. J. Pain Res. 13: 1335–1347.
Canli, T. and Lesch, K. P. (2007). Long story short: the serotonin transporter in emotion regulation and social cognition. Nat. Neurosci. 10(9): 1103–1109.
Cole, M. W., Bassett, D. S., Power, J. D., Braver, T. S. and Petersen, S. E. (2014). Intrinsic and task-evoked network architectures of the human brain. Neuron 83(1): 238–251.
Demori, I., Piccinno, T., Saverino, D., Luzzo, E., Ottoboni, S., Serpico, D., Chiera, M. and Giuria, R. (2021). Effects of winter sea bathing on psychoneuroendocrinoimmunological parameters. Explore 17(2): 122–126.
Godoy, L. D., Rossignoli, M. T., Delfino-Pereira, P., Garcia-Cairasco, N. and de Lima Umeoka, E. H. (2018). A Comprehensive Overview on Stress Neurobiology: Basic Concepts and Clinical Implications. Front. Behav. Neurosci. 12: e00127.
Griffiths, S. and Turner, Y. (2022). Trends in Outdoor Swimming. Second Edition. 2022. Available online: https://www.outdoorswimmershop.com/products/outdoor-swimmer-trends-report-22 (accessed on 18 December 2022).
Grouper, H., Löffler, M., Flor, H., Eisenberg, E. and Pud, D. (2022). Increased functional connectivity between limbic brain areas in healthy individuals with high versus low sensitivity to cold pain: A resting state fMRI study. PLoS One 17(4): e0267170.
Gu, S., Wang, W., Wang, F. and Huang, J. H. (2016). Neuromodulator and Emotion Biomarker for Stress Induced Mental Disorders. Neural Plast. 2016: 1–6.
Hirvonen, J., Lindeman, S., Joukamaa, M. and Huttunen, P. (2002). Plasma catecholamines, serotonin and their metabolites and beta-endorphin of winter swimmers during one winter. Possible correlations to psychological traits. Int. J. Circumpolar Health 61(4): 363–372.
Huttunen, P., Kokko, L. and Ylijukuri, V. (2004). Winter swimming improves general well-being. Int. J. Circumpolar Health 63(2): 140–144.
Jarrahi, B., Martucci, K. T., Nilakantan, A. S. and Mackey, S. (2017). Investigating the BOLD spectral power of the intrinsic connectivity networks in fibromyalgia patients: A resting-state fMRI study. Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference, 2017: 497–500.
Kelly, J. S. and Bird, E. (2022). Improved mood following a single immersion in cold water. Lifestyle Medicine 3(1): e53.
Knechtle, B., Waśkiewicz, Z., Sousa, C. V., Hill, L. and Nikolaidis, P. T. (2020). Cold Water Swimming—Benefits and Risks: A Narrative Review. Int. J. Environ. Res. Public Health 17(23): 8984.
Lindeman, S., Hirvonen, J. and Joukamaa, M. (2002). Neurotic psychopathology and alexithymia among winter swimmers and controls--a prospective study. Int J Circumpolar Health.61(2): 123–130.
Massey, H., Gorczynski, P., Harper, C. M., Sansom, L., McEwan, K., Yankouskaya, A. and Denton, H. (2022). Perceived Impact of Outdoor Swimming on Health: Web-Based Survey. Interact. J. Med. Res. 11(1): e25589.
Massey, H., Kandala, N., Davis, C., Harper, M., Gorczynski, P. and Denton, H. (2020). Mood and well‐being of novice open water swimmers and controls during an introductory outdoor swimming programme: A feasibility study. Lifestyle Medicine 1(2): e12.
Tipton, M. J. (1989). The Initial Responses to Cold-Water Immersion in Man. Clin. Sci. 77(6): 581–588.
Tipton, M. J., Collier, N., Massey, H., Corbett, J. and Harper, M. (2017). Cold water immersion: kill or cure? Exp. Physiol. 102(11): 1335–1355.
Watson, D., Clark, L. A. and Tellegen, A. (1988). Development and validation of brief measures of positive and negative affect: The PANAS scales. J Pers Soc Psychol. 54(6): 1063–1070.
Whitfield-Gabrieli, S. and Nieto-Castanon, A. (2012). Conn: a functional connectivity toolbox for correlated and anticorrelated brain networks. Brain connectivity 2(3): 125–141.