Breathwork and Mindfulness Interventions on Psychological Flexibility: A 4-Arm RCT (BREATH-4)

Effect of Brief Breathwork and Mindfulness Interventions on Psychological Flexibility and Physiological Variables in Spanish-Speaking University Students: Protocol of a Four-Arm Randomized Controlled Trial

This four-arm, single-blind randomized controlled trial examines the effects of four brief daily 5-minute practices over 28 days on psychological flexibility and physiological variables in Spanish-speaking adults. Participants will be randomly assigned to one of four groups: Box Breathing, Cyclic Sighing, Mindfulness focused on exhalation with the syllable "AH", or Conscious Cyclic Sighing. Outcomes include state and trait anxiety (STAI), positive and negative affect (PANAS), psychological flexibility (MPFI-24, Psy-Flex), and physiological variables (heart rate, respiratory rate, heart rate variability). Assessments are conducted at baseline, daily during the 28-day intervention, at post-intervention (day 31), and at 2-month follow-up.

Study Overview

• Status: Not yet recruiting
• Conditions: Stress; Anxiety; Psychological Flexibility
• Intervention / Treatment: Behavioral: Box Breathing; Behavioral: Cyclic Sighing; Behavioral: Mindfulness AH Exhalation; Behavioral: Mindful Cyclic Sighing

• Study Type: Interventional
• Enrollment (Estimated): 240
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Isaac Carmona-Rincón, PhD; Phone Number: +34 618 187 325; Email: isaac.carmona@professor.universidadviu.com

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

• Age between 18 and 65 years (inclusive)
• Native Spanish speaker or fluent in Spanish (sufficient to understand audio instructions and complete psychometric questionnaires)
• Stable internet access and an electronic device (smartphone, tablet, or computer)
• Ability to provide informed consent and commitment to the 31-day protocol and 2-month follow-up

Exclusion Criteria:

• Active diagnosis of severe or chronic respiratory disease that prevents safe performance of breathing exercises (e.g., advanced COPD, severe uncontrolled asthma, cystic fibrosis)
• Current diagnosis of severe psychiatric disorder (e.g., psychotic spectrum disorders, acute-phase bipolar disorder, severe major depression with suicidal risk)
• Any acute medical condition contraindicated for voluntary alteration of breathing patterns

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Single

Number of Arms

4

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Box Breathing; Daily 5-minute box breathing with individualized timing based on a CO2 tolerance test. Participants perform a maximal exhale and record the duration; this determines their personal inhale/hold/exhale/hold intervals (3-4s, 5-6s, or 8-10s). Equal-phase cyclic breathing with exclusive focus on mechanical control and counting.	Behavioral: Box Breathing; Individualized box breathing based on CO2 tolerance test. Participants perform a maximal exhale and record the duration (CO2 discard time) to determine their personal cycle interval: 3-4s (0-20s discard), 5-6s (25-45s discard), or 8-10s (50-75s+ discard). Equal-phase cycles of inhale/hold/exhale/hold repeated for 5 minutes daily. Focus on mechanical breath control only.
Experimental: Cyclic Sighing; Daily 5-minute cyclic sighing. Participants inhale slowly through the nose, then take a second shorter nasal inhale to maximally fill the lungs, followed by a slow, complete oral exhalation. This double-inhale/long-exhale pattern is repeated continuously for 5 minutes. No mindfulness or attentional instructions are given.	Behavioral: Cyclic Sighing; Double nasal inhalation (slow deep inhale + short top-up inhale to maximal lung capacity) followed by slow complete oral exhalation. Repeated continuously for 5 minutes daily. No attentional instructions.
Experimental: Mindfulness AH Exhalation; Daily 5-minute focused-attention mindfulness practice. Participants sit or lie down, close their eyes, and direct attention to the forehead region between the eyes. On each exhale, they softly vocalize the syllable "AH" aloud (gentle, unforced audible sound). When distracted, they use the internal label "thinking-thinking" and redirect attention to the breath and then to the forehead region. Practice continues for 5 minutes.	Behavioral: Mindfulness AH Exhalation; Focused-attention practice with eyes closed. Attention directed to forehead region between the eyes. Each exhalation accompanied by soft audible vocalization of syllable "AH". Distractions managed with internal label "thinking-thinking" followed by attention redirection. Practiced daily for 5 minutes.
Experimental: Mindful Cyclic Sighing; Daily 5-minute hybrid practice combining cyclic sighing mechanics with mindfulness instructions. Participants inhale slowly through the nose, take a second shorter nasal inhale to maximally fill the lungs, then exhale slowly while softly vocalizing the syllable "AH" aloud (gentle, unforced). Attention is directed to the breath and the sensation of release during exhalation. When distracted, participants use the internal label "thinking-thinking" and redirect attention back to the breath. Practice continues for 5 minutes.	Behavioral: Mindful Cyclic Sighing; Hybrid practice: double nasal inhalation (cyclic sighing mechanics) combined with slow exhalation vocalized as soft audible "AH". Mindfulness instructions include attention to breath sensations and use of "thinking-thinking" label upon distraction. Practiced daily for 5 minutes.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
State Anxiety (STAI-E)	State anxiety measured with the State-Trait Anxiety Inventory - State subscale (STAI-E). 20 items rated on a 4-point Likert scale (0-3). Higher scores indicate greater anxiety. Spanish validated version (Guillen-Riquelme & Buela-Casal, 2011).	Baseline (Day 0), daily pre- and post-practice (Days 1-28), post-intervention (Day 31), and 2-month follow-up

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Positive and Negative Affect (PANAS)	Positive and negative affect measured with the Positive and Negative Affect Schedule (PANAS). 20 items rated on a 5-point Likert scale. Two subscales: Positive Affect (PA) and Negative Affect (NA). Spanish validated version (Lopez-Gomez et al., 2015).	Baseline (Day 0), daily pre- and post-practice (Days 1-28), post-intervention (Day 31), and 2-month follow-up
Psychological Flexibility (MPFI-24)	Multidimensional psychological flexibility and inflexibility measured with the Multidimensional Psychological Flexibility Inventory short form (MPFI-24). 24 items rated on a 6-point Likert scale assessing 12 Hexaflex processes. Spanish validated version (Navarrete et al., 2024).	Baseline (Day 0), post-intervention (Day 31), and 2-month follow-up
Psychological Flexibility (Psy-Flex)	Unified psychological flexibility measured with the Psy-Flex scale. 6 items rated on a 5-point Likert scale. A modified daily version will be used during the intervention period. Spanish validated version (Navarrete et al., 2024).	Baseline (Day 0), daily (Days 1-28), post-intervention (Day 31), and 2-month follow-up
Trait Anxiety (STAI-R)	Trait anxiety measured with the State-Trait Anxiety Inventory - Trait subscale (STAI-R). 20 items rated on a 4-point Likert scale (0-3). Higher scores indicate greater trait anxiety. Spanish validated version (Guillen-Riquelme & Buela-Casal, 2011).	Baseline (Day 0), post-intervention (Day 31), and 2-month follow-up

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Resting Heart Rate (RHR)	Resting heart rate in beats per minute (bpm) obtained from participants' personal wearable devices (smartwatch/fitness tracker). Change calculated as the slope of a linear regression fitted to daily values over the 28-day period.	Daily (Days 1-28)
Heart Rate Variability (HRV)	Heart rate variability obtained from participants' personal wearable devices during deep sleep. Change calculated as the slope of a linear regression fitted to daily values over the 28-day period. Only available for participants with compatible devices.	Daily (Days 1-28)
Respiratory Rate (RR)	Respiratory rate in breaths per minute obtained from participants' personal wearable devices. Change calculated as the slope of a linear regression fitted to daily values over the 28-day period. Only available for participants with compatible devices.	Daily (Days 1-28)

Collaborators and Investigators

• Sponsor: Valencian International University (VIU)
• Investigators: Principal Investigator: Isaac Carmona-Rincón, PhD, Valencian International University (VIU)

Publications and helpful links

General Publications

• Balban MY, Neri E, Kogon MM, Weed L, Nouriani B, Jo B, Holl G, Zeitzer JM, Spiegel D, Huberman AD. Brief structured respiration practices enhance mood and reduce physiological arousal. Cell Rep Med. 2023 Jan 17;4(1):100895. doi: 10.1016/j.xcrm.2022.100895. Epub 2023 Jan 10.
• Chan AW, Boutron I, Hopewell S, Moher D, Schulz KF, Collins GS, Tunn R, Aggarwal R, Berkwits M, Berlin JA, Bhandari N, Butcher NJ, Campbell MK, Chidebe RCW, Elbourne DR, Farmer AJ, Fergusson DA, Golub RM, Goodman SN, Hoffmann TC, Ioannidis JPA, Kahan BC, Knowles RL, Lamb SE, Lewis S, Loder E, Offringa M, Ravaud P, Richards DP, Rockhold FW, Schriger DL, Siegfried NL, Staniszewska S, Taylor RS, Thabane L, Torgerson DJ, Vohra S, White IR, Hrobjartsson A. SPIRIT 2025 statement: updated guideline for protocols of randomized trials. Nat Med. 2025 Jun;31(6):1784-1792. doi: 10.1038/s41591-025-03668-w. Epub 2025 Apr 29.

Study record dates

Study Major Dates

• Study Start (Estimated): January 1, 2027
• Primary Completion (Estimated): March 1, 2027
• Study Completion (Estimated): May 1, 2027

Study Registration Dates

• First Submitted: May 27, 2026
• First Submitted That Met QC Criteria: May 27, 2026
• First Posted (Actual): June 2, 2026

Study Record Updates

• Last Update Posted (Actual): June 2, 2026
• Last Update Submitted That Met QC Criteria: May 27, 2026
• Last Verified: May 1, 2026

More Information

Terms related to this study

• Keywords: randomized controlled trial; mindfulness; heart rate variability; Spanish-speaking; box breathing; breathworks; cyclic sighing
• Additional Relevant MeSH Terms: Mental Disorders; Anxiety Disorders
• Other Study ID Numbers: VIU-BREATH-RCT-2026

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: Anonymized individual participant data (IPD) and the data dictionary will be made available upon reasonable request to the principal investigator (isaac.carmona@professor.universidadviu.com). Requests will be evaluated on a case-by-case basis.
• IPD Sharing Time Frame: Beginning 6 months after publication of primary results, available for 5 years
• IPD Sharing Access Criteria: Researchers who provide a methodologically sound proposal. Data shared under a data sharing agreement ensuring confidentiality and appropriate use.
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; SAP; ANALYTIC_CODE

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No