Changes in Heart Rate Variability, Heart Rate, Blood Pressure, and Mood After Walking in Green Spaces. A Comparison of Environments With Healthy Adults.
Phase: NA
Conditions: Healthy Subjects (HS), Forest Therapy, Autonomic Nervous System Activity, Mood
Sponsor: University of Vigo

Title: Effects of walking in different tree-dominated environments on autonomic 
response in adults over 50: a randomized crossover trial 
 
NCT07114146  
Date: April 14, 2026 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

1. Study Design 
This study was designed as a randomized crossover field trial to evaluate the 
physiological and psychological responses associated with exposure to different 
environments during a leisurely walk. 
Each participant was exposed to a total of four experimental conditions, 
consisting of three tree-lined outdoor environments with different environmental 
and ecosystem characteristics (A, B, and C) and a control condition in an indoor 
environment (D).  
Each condition constituted an independent period of the crossover design and 
was conducted on different days. 
The research staff was responsible for implementing the protocol, following a 
prior internal training process aimed at ensuring the standardization of 
procedures, the proper handling of devices, and consistency in the instructions 
provided to participants. Each group of participants was accompanied at all times 
by two researchers. 
2. Intervention Sequences and Randomization 
Randomization was applied to the assignment of participants to complete 
exposure sequences, not to individual conditions. 
Three intervention sequences were defined: 
• 
ABCD 
• 
BCAD 
• 
CABD 
Assignment was performed using a computerized procedure with R (randomizeR 
package), with a 1:1:1 ratio between sequences. 
Each participant was assigned to a sequence after signing the informed consent 
form and being assigned a unique identifier. 
Condition D (in-house control) was always performed in the last period, for 
methodological and logistical reasons related to the standardization of walking 
speed on the treadmill. This aspect was considered in the analysis and 
interpretation of the results as a possible period effect. 
3. Blinding 
It was not possible to blind either the participants or the staff responsible for the 
intervention. 
However: 
• 
the participants were unaware of the assigned sequence 

• 
they were not informed of the nature of the conditions or the existence of 
a control condition 
Three individuals outside the research team participated exclusively in the blood 
pressure and heart rate measurements for M1 and M4. These evaluators 
remained blinded to the setting of each session through physical and functional 
separation from the rest of the team and through the exclusive use of the 
participant’s identifier during data collection. No breaches of blinding were 
recorded. 
4. Participants 
The study included 16 healthy adult participants aged 50 years or older, residing 
in the vicinity of the study area and with sufficient functional capacity to complete 
a leisurely 3-km walk independently and safely. 
Inclusion Criteria 
Participants had to meet all of the following criteria: 
1. Age 50 years or older. 
2. Ability to independently perform a low-intensity walk. 
3. No prior diagnosis of serious cardiovascular, respiratory, neurological, 
musculoskeletal, or psychiatric conditions that could interfere with 
participation or the interpretation of results. 
4. No severe allergies related to exposure to outdoor environments during 
the study period. 
5. Commitment to completing the four planned experimental sessions. 
6. Attendance at an in-person informational meeting prior to the start of the 
study. 
7. Signing of a written informed consent form prior to any procedure. 
8. Availability of a mobile device compatible with the Kubios HRV Daily 
Readiness app and equipped with Bluetooth connectivity. 
The following were excluded: 
1. Individuals with regular use of narcotic or non-narcotic drugs. 
2. Pregnant women. 
3. Individuals with any clinical condition or functional limitation that could 
compromise safety during the protocol or the validity of the measurements. 
 
Recruitment was conducted via self-selection, through advertisements posted on 
social media, posters on the university campus, and the university’s institutional 
channels. 
Interested individuals initially completed an online screening form. Those who 
met the preliminary criteria were invited to an in-person informational meeting 

where eligibility was verified, questions were answered, and inclusion was 
formalized through the signing of the informed consent form. 
Recruitment took place between July 28 and September 4, 2025. The in-person 
informational meeting was held on September 4, 2025, and the experimental 
sessions took place between September 9 and 22, 2025. 
5. Ethical Considerations 
The study was approved by the Ethics Committee of the University of León 
(Spain) under code ETICA-ULE-087-2025, with an approval date of July 25, 
2025. 
The study was registered on ClinicalTrials.gov with identifier NCT07114146 on 
July 27, 2025. 
The research was conducted in accordance with the Declaration of Helsinki and 
applicable regulations regarding research involving human subjects and data 
protection. 
All participants received verbal and written information regarding the study’s 
objectives, procedures, risks, benefits, and their right to withdraw at any time 
without penalty, and signed the corresponding informed consent form before the 
start of any study procedures. 
6. Study Conditions 
The study was conducted in the city of Pontevedra (Spain), in a peri-urban setting 
near the A Xunqueira University Campus. 
Four experimental conditions were evaluated: 
• 
Setting A: Peri-urban wooded area located on the outskirts of the 
University Campus 
• 
Environment B: A humid semi-natural environment located in a marshland 
area, dominated by marsh and halophilic vegetation adapted to saturated 
soils 
• 
Environment C: Peri-urban riparian forest 
• 
Environment D (indoor control): Enclosed room with white walls and a 
treadmill. 
The first three corresponded to forested outdoor environments with distinct 
environmental and ecosystem characteristics. The fourth corresponded to an 
indoor control condition in the laboratory. 
7. Intervention 
All participants walked 3 km under the four experimental conditions. 

In outdoor conditions A, B, and C, the walk was performed at a self-paced pace, 
with the express instruction to maintain a calm and comfortable pace, without 
imposing a target speed. 
In indoor condition D, the walk was performed on a BH HiPower SIRIUS 3.0 
treadmill at a preset speed corresponding to the individual average speed 
obtained by each participant in condition A, calculated in km/h based on the time 
taken to cover 3 km. This speed could be adjusted downward for safety or 
tolerance reasons, at the discretion of the principal investigator. 
The three outdoor routes had low and comparable gradients, without steep 
inclines, with the aim of promoting a consistent physical workload. 
8. Study Procedure 
The three outdoor sessions took place in the morning on September 9, 12, and 
15, 2025. Participants were scheduled to arrive at 8:45 a.m. at the Faculty of 
Physical Therapy on the University Campus. The estimated duration of each 
outdoor session was until approximately 2:00 p.m. 
The indoor condition took place on September 18, 19, or 22, 2025, during time 
slots adapted to the availability of the participants and the center, with an 
approximate duration of 2 hours per participant. 
8.1 Procedure under outdoor conditions 
Upon arrival at the meeting point, measurement M1 was performed. 
Subsequently, participants were transported as a group to the corresponding 
location. 
• 
For conditions B and C, transportation was by bus. 
• 
For condition A, and in order to maintain comparable waiting and rest 
times, participants remained seated for a time similar to the bus ride. 
• 
After the walk, comparable return or waiting times were also applied 
depending on the condition. 
Participants began the walk in staggered intervals every 5 minutes to facilitate 
individual completion of the route. 
8.2 Procedure for the indoor condition 
In condition D, participants arrived individually at the School of Physical Therapy 
at a predetermined time. The same general phases of the protocol were 
replicated, but without the waiting time associated with the group outdoor walks. 
 
 
 

9. Study variables 
The following were defined as prespecified study outcomes: 
Variables derived from HRV measurement  
• 
RR interval 
• 
RMSSD 
• 
SDNN 
• 
HF power 
• 
LF power 
Cardiovascular variables 
• 
Heart rate 
• 
Systolic blood pressure 
• 
Diastolic blood pressure 
Psychological variables 
• 
POMS 
• 
TMD 
Perceptual variables 
• 
Perceived environmental quality (A, B, C) 
10. Measurements 
10.1 Heart rate variability (all variables) 
The heart rate signal was recorded using a Polar H10 chest strap connected via 
Bluetooth to the Kubios HRV Daily Readiness mobile app, with subsequent 
export of the data to Kubios HRV Scientific version 4.1.2. 
The signal was recorded continuously starting 5 minutes before the start of the 
walk, throughout the entire 3-km walk, and up to 5 minutes after its completion. 
The following variables were obtained from the continuous recording: 
• 
RR interval 
• 
RMSSD 
• 
SDNN 
• 
HF power  
• 
LF power  
The unit of measurement for RR interval, RMSSD, and SDNN was milliseconds 
(ms). The HF and LF spectral components were expressed in ms². 

The chest strap was placed according to the manufacturer’s recommendations, 
after moistening the electrodes and adjusting the elastic band to fit the chest. The 
start and end of the recording were performed by members of the research team. 
Signal processing was performed using automatic mean-level noise detection 
and automatic beat correction. The quality criterion was set to exclude recordings 
with more than 5% artifacts or corrected beats. 
10.2 Blood Pressure and Heart Rate 
Systolic blood pressure, diastolic blood pressure, and heart rate were measured 
using a clinically validated Omron M2 automatic sphygmomanometer. 
Measurements were taken on the left arm, with the participant in a seated 
position, back supported, feet flat on the floor, and arm at heart level. Before each 
measurement, the participant rested in a seated position for 5 minutes. 
At each time point, three consecutive readings were taken, one minute apart. The 
first reading was systematically discarded, and the final value was calculated as 
the average of the second and third readings. 
Blood pressure was expressed in mmHg and heart rate in beats per minute. 
10.3 Mood and Total Mood Disturbance  
To measure mood, the 30-item short form of the POMS scale validated in Spanish 
was used (Andrade et al., 2013). The Profile of Mood States is a questionnaire 
based on a multidimensional conception of mood, designed to obtain information 
on fluctuations in emotional states across different contexts. In our study, we used 
the short version of the POMS in Spanish, which contains 30 items comprising 6 
factors, one for each of the main mood factors included in the POMS. Four of 
these factors are negative in nature—Anger, Fatigue, Tension, and Depressed 
Mood—and two are positive: Vigor and Friendliness. It is presented as a Likert-
type questionnaire with 5 options for each factor, to which values between 0 
(None) and 4 (Very Much) are assigned. 
Another piece of data obtained from the POMS is the total score, or Total Mood 
Disturbance (TMD), calculated by summing all the values obtained for each 
factor, with the exception of the two positive factors, which are subtracted. 
10.4 Perceived Environmental Quality  
A proprietary instrument called the “Perceived Environmental Quality Scale of the 
Forest Environment,” whose main objective is to quantify the extent to which the 
perceived attributes and characteristics of forest environments affect a person’s 
experience when coming into contact with the evaluated space.  
The scale consists of 41 questions. Thirty-nine of these are scored on a 1-to-5-
point Likert scale, where 1 = “none” and 5 = “a lot”; The last two questions on the 

scale are referred to as “criterion questions” and are scored on a scale of 1 to 10 
points, where 1 = “detestable – none” and 10 = “outstanding – very much.” 
11. Physiological Measurement Time Points 
Systolic blood pressure, diastolic blood pressure, and heart rate were measured 
at four time points during each period: 
• 
M1: arrival at the meeting point at the School of Physical Therapy on the 
University Campus. 
• 
M2: 5 minutes before the start of the walk in the corresponding 
environment. 
• 
M3: 5 minutes after the walk ended, in the corresponding environment. 
• 
M4: after returning to the meeting point on the University Campus. 
The continuous signal for RR interval and HRV was recorded from 5 minutes 
before the walk until 5 minutes after its completion. 
Mood was assessed using the Profile of Mood States (POMS) questionnaire. The 
POMS was administered at two points during each session: 
• 
immediately after M1 
• 
immediately after M3 
The Total Mood Disturbance (TMD) index was subsequently calculated based on 
these scores. 
Perceived 
environmental 
quality 
was 
assessed 
using 
the 
Perceived 
Environmental Quality Scale under outdoor conditions A, B, and C. This scale 
was administered after M3, along with the POMS questionnaire. This 
measurement was not performed in the indoor control condition. 
11. Environmental and Microclimatic Variables 
Meteorological and microclimatic data were recorded each study day between 
9:00 and 9:30 a.m., before the participants arrived at the environments. 
In conditions A, B, and C, meteorological variables were obtained from the 
Pontevedra-Campolongo station. Noise levels were recorded using a CESVA 
SC-30 digital sound level meter at the start and end points of the walks. 
In condition D, temperature and relative humidity were recorded at the midpoint 
of the room using a Potermic 72000 multifunction meter with real-time monitoring. 
12. Statistical Analysis 
The data will be analyzed using statistical models appropriate for repeated-
measures designs in a crossover context, taking into account the intra-individual 
dependence of the observations. 

The analysis may include: 
• 
longitudinal modeling of continuous variables 
• 
comparison between experimental conditions 
• 
evaluation of the temporal evolution of the variables 
• 
adjustment for individual covariates (age, sex, weight, and height) 
• 
inclusion of random effects at the participant level 
Mixed linear models or other equivalent approaches may be used depending on 
the nature of each variable. 
Analyses will be performed using the statistical software R. 
13. Final considerations regarding the scope of the protocol 
This protocol describes the entire study, including all variables collected as part 
of the trial. The results may be published in one or more scientific journals, without 
this altering the unified nature of the study or the pre-specified nature of the 
variables and procedures described herein.