INTRODUCTION
Structural and functional changes in the musculoskeletal system associated with aging paradoxically reduce quality of life, despite the increase in life expectancy (D’Onofrio et al., 2023). Sarcopenia, characterized by a decline in skeletal muscle mass and strength, is accompanied by a relative increase in adipose tissue, which induces inflammatory markers in various tissues and organs (Wang et al., 2017). These changes not only alter blood components but also impair immune cell function (Heo et al., 2024), potentially leading to a shortened lifespan. The decline in muscle mass among older adults is driven by alterations in the production and sensitivity of various hormones, leading to reduced muscle mass and increased fat accumulation, which ultimately induces changes not only in body composition but also in blood constituents (Brun, 2002).
Recent studies have reported that regular exercise improves both the structure and function of skeletal muscle not only in healthy young individuals but also in older adults (Distefano and Goodpaster, 2018). Regular exercise plays a crucial role in preserving skeletal muscle mass by promoting the secretion of signalling proteins that support erythrocyte’s function and maintain the normal activity of immune cells (Bruunsgaard and Pedersen, 2000). In addition to exercise, numerous studies have reported that various substances released in forest environments—such as phytoncides—contribute to enhancing immune cell function in humans (Park et al., 2023; Tsao et al., 2018). Building upon the aforementioned benefits of regular exercise, many researchers have long emphasized forest hiking (FH) as a form of physical activity (PA) capable of inducing substantial physiological and psychological adaptations in humans. Natural environments characterized by scenic landscapes, harmony with nature, and balanced ecosystems enrich the hiking experience (Li, 2022). In Korea, distinct seasonal variations, clean water, and fresh air, combined with numerous temples and cultural resources, create an ideal setting for age-friendly hiking. Moreover, many Korean mountains feature relatively gentle slopes, making FH a suitable and safe activity for older adults. With the growing embrace of eco-friendly lifestyles and increasing interest in nature-based sports in modern society, FH is emerging as a meaningful and beneficial leisure activity for the elderly (Catissi et al., 2024).
Mountains are widely recognized for their diverse health benefits, leading to the emergence of the term forest healing or forest therapy (Heckmann et al., 2024). This concept encompasses therapeutic and restorative interventions carried out within mountain forests, making use of their unique physical and environmental features to support health recovery, maintenance, and improvement. The forest environment fosters both psychological and physiological stability, primarily through bioactive compounds released by trees, with phytoncides being the most prominent (Li, 2022). Multiple studies have shown that phytoncides decrease stress hormone levels, thereby facilitating the activation and proliferation of natural killer (NK) cells (Masuo et al., 2021). Heo et al. (2024) further demonstrated a significant postintervention increase in NK-cell subsets within the phytoncide group, suggesting that phytoncide inhalation not only alleviates stress but also enhances immune function. Consistent with these findings, Li et al. (2006) reported that phytoncides directly stimulate NK-cell proliferation. Likewise, Li (2010) observed that even a brief two-day forest bathing experience significantly elevated NK-cell activity, with the immunological benefits sustained for up to 30 days.
Considering previous findings, FH may provide substantially greater benefits for older adults compared to walking in urban or non-PA environments. Unlike routine flat-ground activities, hiking on sloped forest trails can help preserve muscle function and potentially induce hypertrophic adaptations, thereby contributing to the maintenance or enhancement of both immune cell activity and hematological function. Taken together, these physiological effects suggest that FH may serve as a particularly beneficial form of exercise for older adults. However, the effects of FH on muscle function in this population remain insufficiently studied. To date, no research has examined whether FH improves muscle function by directly measuring parameters such as intramuscular oxygen utilization or saturation. Moreover, little is known about how regular FH influences hematological components and immune cell profiles in older adults. Therefore, the present study aimed to investigate the impact of FH on skeletal muscle oxygen saturation (SmO2) and to examine subsequent changes in muscle function, erythrocyte-related variables, and immune cell subsets following regular participation in this activity.
MATERIALS AND METHODS
Study design
This study was designed as a pilot, prospective, double-blind, randomized controlled trial. Participants were residents of two senior living communities who had neither engaged in regular exercise nor participated in forest walking prior to enrolment. The intervention lasted for 4 weeks, and outcomes included changes in muscle fitness variables and blood components, which were assessed at baseline and after completion of the hiking program. For the control (CON) group, participants did not engage in FH and resided only within the senior tower. The study protocol was reviewed and approved by the Hanseo University Institutional Review Board (approval number: HS23-08-02). The trial was retrospectively registered with the Clinical Research Information Service under the Korea Disease Control and Prevention Agency (registration number: KCT0008712). All procedures complied with the ethical principles of the Declaration of Helsinki (2013).
Participants
Older adults residing in two Seniors Towers in Seoul, Korea, were recruited for this study. Eligible participants were community-dwelling individuals aged 66–78 years who had no physical limitations affecting hiking ability, did not consume alcohol, and had not engaged in regular PA or hiking during the previous 6 months. Before enrolment, all participants underwent a physical examination at Seoul Songdo Hospital, which included demographic assessments, blood sampling, body composition analysis, and muscle function testing. Participants were randomly allocated to one of two groups. The FH group (n=30) engaged in hiking twice weekly for 2 hr per session over a 4-week period, coinciding with peak phytoncide levels in the Gangwon-do region. The CON group (n=30) did not receive an intervention; however, SmO2 of the vastus lateralis (VL) was measured in both groups concurrently with the hiking sessions. Exclusion criteria included recent weight-loss treatments, use of medications affecting body composition, or surgical procedures within the previous year. Individuals with a history of cardiovascular or cerebrovascular disease, cancer, or psychiatric disorders were also excluded.
A two-way repeated-measures analysis of variance (ANOVA) was selected as the primary research design. Sample size estimation was performed using the G*Power program with the following parameters: effect size f=0.25, α=0.05, statistical power (1–β)= 0.95, two groups, and two measurement points. The analysis indicated a minimum requirement of 54 participants (Kang, 2021). To account for an anticipated 20% dropout rate (Sedgwick, 2013), the recruitment target was set at 64. Ultimately, 85 individuals were recruited from two independent facilities to minimize cross-group contamination. Of these, 15 were excluded due to ineligibility or personal reasons, leaving 70 participants who completed demographic and baseline assessments. Randomization was conducted via a tag-selection method by two independent representatives, with one facility allocated to the CON group and the other to the FH group, under the supervision of the principal investigator. Each group initially comprised 35 participants. During the intervention, three participants in the CON group had incomplete SmO2 measurements and two had missing data, yielding 30 participants for the final analysis. In the FH group, two participants withdrew and three had missing data, also resulting in 30 participants in the final analysis as shown in Fig. 1.
FH intervention
FH was conducted in the Osaek area of Seoraksan National Park (Yangyang-gun, Gangwon-do, Korea). Following established recommendations for older adults’ physical capacities (Piercy and Troiano, 2018; Taylor et al., 2004), a course was selected to provide a light-to-high intensity workload. Each session began and ended with 10–15 min of stretching. The intervention was delivered over 4 weeks, twice weekly (Tuesdays and Fridays), with participants instructed to complete a round-trip hike within 120 min. Pilot testing of the course indicated a cumulative elevation gain of 107 m, an average walking speed of 3.1–3.8 km/hr, and a step count of 6,700–7,100 per session. Exercise intensity was prescribed at 40%–70% of each participant’s maximal oxygen uptake (VO2max), determined via a treadmill-based graded exercise test. To maintain this target, individualized heart rate zones were pre-programmed into smartwatches worn during hiking. Subjective effort was assessed every 10–20 min using Borg 10-point rating of perceived exertion scale (Borg, 1982). If exertion exceeded 80% of VO2max, participants rested for 2–3 min; these breaks were not included in the 120-min hiking duration.
Muscle SmO2 and VO2max measures
In the FH group during hiking sessions and in the CON group during daily living, activity volume was assessed using a Moxy Monitor (Muscle Oxygen Monitor, Fortiori Design LLC, USA). This device applies near-infrared spectroscopy to provide SmO2 values on a 0%–100% scale with established accuracy. The default sampling rate cycles through wavelengths 80 times every 2 sec, yielding an output frequency of 0.5 Hz (Feldmann et al., 2019). SmO2 values were selectively extracted from the muscle tissue layer. During measurements, the monitor was affixed to the VL using hypoallergenic, low-adhesive patches suitable for sensitive skin. To optimize accuracy, a detector response matrix was generated through ray-tracing simulations based on the optical properties of biological tissue, incorporating expected SmO2 ranges and published absorption spectra of key chromophores within each tissue layer (Feldmann et al., 2019).
Hiking intensity was prescribed based on VO2max, assessed using a graded exercise test with a cardiopulmonary exercise testing system (Quark CPET, Cosmed, Italy). The modified Bruce protocol, validated for older adults (Kim et al., 2025), was applied on a treadmill ergometer (T150, HP/Cosmos, Germany) with continuous electrocardiographic and blood pressure monitoring. Tests were performed under medical supervision and terminated according to standard safety criteria outlined by the American College of Sports Medicine Position Stand (1998).
Demographic data and daily dietary/activity measures
Demographic variables included age, sex, family living arrangement, chronic disease status, and level of social activity. Sex was coded as 1 for men and 2 for women. Living arrangement was coded as 1 for living alone and 2 for living with a spouse or family, with presence coded as 1 and absence as 0. Chronic disease status was recorded as multiple responses, including none, osteoporosis, overweight, obesity, otorhinolaryngologic disorders, endocrine disorders, urological disorders, dyslipidemia, liver disease, spinal deformity, hypertension, diabetes, gastrointestinal disease, and arthritis. Social activity was assessed on a 5-point scale (1=very good; 5=very poor). Family history of heart disease and smoking status were each coded as 1 (present) or 0 (absent).
Daily caloric intake was assessed using meal plans designed by a registered dietitian, which included macronutrients and micronutrients. A single researcher entered the dietary data into the CAN-Pro 5.0 program (http://canpro5.kns.or.kr; Korean Nutrition Society, Korea) to record and analyze the daily intake of all participants. Daily PA levels were estimated using the International PA Questionnaire–Short Form (Chun, 2012). Daily caloric intake and PA levels were recorded daily from the beginning to the end of the intervention. These data were aggregated on a monthly basis and averaged for comparative analysis between the pre- and postintervention periods.
Body composition measure
It was conducted at 7:00 a.m., the scheduled wake-up time, with participants reporting to the designated testing area in a predetermined sequence. Body composition parameters were assessed using the InBody 720 device (Biospace, Korea). Body mass index was calculated as weight in kilograms divided by height in meters squared.
Thigh muscle function measure
Muscle contractile properties were evaluated using a tensiomyography (TMG) system (TMG100, TMG-BMC Ltd., Slovenia), which consists of an electrical stimulator, a displacement sensor, and analysis software. The system provides five parameters: maximal displacement (Dm), delay time (Td), contraction time (Tc), sustain time (Ts), and relaxation time (Tr). For measurement, the sensor was placed on the belly of the target muscle with surface electrodes positioned 2–3 cm away along the fiber direction. Electrical stimulation was initiated at 20 mA and increased in 10-mA increments until maximal displacement was reached. To prevent potentiation and fatigue, stimulations were applied at 10-sec intervals. The VL and biceps femoris (BF) were selected due to their importance in lower-limb propulsion and shock absorption during hiking. For VL assessment, participants were positioned supine with knees flexed at 120°, while for BF assessment, they were prone with knees flexed at 150°. Prior to testing, participants rested for 5 min to ensure complete muscle relaxation. Tc and Dm were derived from the displacement–time curve (Dahmane et al., 2001; Park et al., 2023).
Blood biomarkers measures
Blood samples were collected from the median cubital vein after a 10-hr overnight fast using three types of BD Vacutainer tubes (BD Biosciences, USA): K2-EDTA tubes for differential blood cell counts, heparin tubes for flow cytometric analysis, and serum tubes. Serum samples were allowed to clot upright for 30–45 min, then centrifuged at 1,000 ×g for 10 min. Approximately 2-mL aliquots of the serum were stored at −80°C for long-term preservation and subsequent analyses. For analying erythrocytes, within 2 hr of collection, samples were analyzed using an automated hematology analyzer (XN-1000, Sysmex Corporation, Japan). Prior to analysis, daily internal quality control was performed with normal and abnormal control materials provided by the manufacturer, and calibration was carried out according to the manufacturer’s instructions. Erythrocytes were counted (×106/μL) using the electrical impedance method, which measures changes in electrical resistance as cells pass through a microaperture. Hemoglobin (Hb) concentration (g/dL) was determined spectrophotometrically (approximately 525–555 nm) using the sodium lauryl sulfate-Hb method, which stabilizes Hb complexes after hemolysis of whole blood. Hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular Hb concentration (MCHC), and red cell distribution width (RDW) were calculated automatically by the analyzer.
To assess cellular immune function, the counts and distribution of key immune cell populations were evaluated. Flow cytometry was performed to quantify the relative and absolute numbers of lymphocyte subsets, including CD56+ NK cells, NK T cells, CD19+ B cells, CD3+ T cells, CD4+ helper T cells, and CD8+ cytotoxic T cells. A 50 μL whole blood sample was stained with fluorescently conjugated monoclonal antibodies: CD56-PE, CD19, CD3-FITC, CD4-BV510, CD8-and PerCP-Cy5.5. Samples were incubated in the dark at room temperature for 15 min, followed by red blood cell lysis using fluorescence-activated cell sorting (FACS) lysing solution for an additional 15 min. Flow cytometric analysis was conducted on a FACS Canto II instrument (BD Biosciences, USA) and data were analyzed using FlowJo software (Treestar, USA). Absolute lymphocyte subset counts were determined with an automated hematology analyzer. Additionally, optical density was measured at 450 nm with wavelength correction at 540 nm using a Tecan Sunrise microplate reader (Tecan Group Ltd., Switzerland), in accordance with protocols (Hulspas et al., 2009; Lee and Jee, 2021).
Statistical analysis
All the data are reported as the means±standard deviations and were analyzed via GraphPad Prism 10.4.1 (USA). The Kolmogorov–Smirnov test was employed to check the normality of the demographic and anthropometric data. For normally distributed data, repeated-measures ANOVA was applied for variable comparisons. For nonnormally distributed data, the Mann–Whitney U-test was used to analyze differences between groups. Intragroup changes were evaluated via the Wilcoxon signed-rank test. A detailed analysis involved calculating the Δ% for each period. Effect sizes (η2) were interpreted as small, moderate, or large on the basis of thresholds of 0.2, 0.5, and 0.8 for parametric measures and 0.1, 0.3, and 0.5 for nonparametric measures, respectively (Fritz et al., 2012). Statistical significance was set at P≤0.05.
RESULTS
Demographic characteristics
As shown in Table 1, a total of 60 participants were analyzed, with 30 allocated to the CON group (20 males, 10 females) and 30 to the FH group (20 males, 10 females). No significant differences were observed between groups in sex distribution, living arrangements, social activity levels, or family history of heart disease, including smoking. Body composition variables were also comparable between groups. However, differences were identified in the prevalence of chronic diseases.
Differences and changes in daily calorie input/output and body composition
Prior to the intervention, mean calorie intake was 1,759.10± 108.41 kcal in the CON group and 1,717.77±154.27 kcal in the FH group. Postintervention, intake was 1,725.17±249.60 kcal and 1,724.13±156.55 kcal, respectively (Z=−0.955, P=0.340, η2=0.012). As illustrated in Fig. 2A, the Δ% in calorie intake decreased by −1.49%±15.98% in the CON group but increased by 1.15%±12.95% in the FH group, showing no significant between-group difference (Z=−0.385, P=0.700, η2=0.008). Baseline PA levels were 412.40±54.54 MET·min/wk in the CON group and 412.40±48.67 MET·min/wk in the FH group (Z=−0.044, P=0.965). Following the intervention, PA levels declined to 396.43±60.56 MET·min/wk in the CON group but markedly increased to 1,191.13±125.87 MET·min/week in the FH group (Z=−6.655, P=0.001, η2=0.944). As shown in Fig. 2B, Δ% PA levels decreased by −1.92%±21.30% in the CON group while increasing by 192.99%±47.71% in the FH group, indicating a significant between-group difference (Z=−6.653, P=0.000, η2= 0.878).
Baseline body weight was 67.26±7.26 kg in the CON group and 68.24±6.72 kg in the FH group. Postintervention, it increased to 69.57±7.63 kg in the CON group but decreased to 65.20± 11.42 kg in the FH group. As shown by the Δ% change, it increased by 3.53%±5.62% in the CON group and decreased by −4.26%± 8.35% in the FH group, resulting in a significant between-group difference (Z=−3.482, P=0.000, η2=0.236). A similar pattern was observed for body mass index. Muscle mass at baseline was 32.95±5.82 kg in the CON group and 32.38±5.66 kg in the FH group. Following the intervention, it declined to 29.81±3.09 kg in the CON group but increased to 35.56±5.15 kg in the FH group. The Δ% change indicated a reduction of −7.61%±13.49% in the CON group and an increase of 10.58%±8.17% in the FH group, producing a significant between-group difference (Z=−4.216, P=0.000, η2=0.407) (Fig. 2C). In contrast, fat mass at baseline was 19.39±3.71 kg in the CON group and 19.04±5.06 kg in the FH group. After the intervention, it increased to 21.62±4.48 kg in the CON group but decreased to 16.84±4.42 kg in the FH group. The Δ% change showed an increase of 13.59%±21.65% in the CON group compared with a decrease of −10.95%±9.44% in the FH group, yielding a significant between-group difference (Z=−5.122, P=0.000, η2=0.358) (Fig. 2D).
Differences and changes in muscle’ contractility in TMG
As presented in Table 2, no significant differences in muscle contractility measured by TMG were observed between the groups before the experiment. After the intervention, the VL in the CON group slowed on both sides, whereas it accelerated in the FH group; however, a significant interaction effect was observed only in the right VL Tc. A similar trend was also noted in the BF. Meanwhile, Dm decreased bilaterally in the VL of the CON group but increased in the FH group, demonstrating a significant interaction effect. Comparable results were also observed in the BF.
Fig. 3 illustrates the Δ% changes in muscle contractility variables between the two groups from pre- to postintervention. The Tc of the left and right VL increased in the CON group by 15.94%± 43.36% and 18.81%±58.91%, respectively, but decreased in the FH group by −6.60%±44.59% and −9.75%±47.77%, respectively, showing significant between-group differences of the right side (Fig. 3A). Similarly, Tc of the left and right BF increased in the CON group by 10.15±53.98% and 11.73±38.90%, respectively, whereas it decreased in the FH group by −7.76%±29.51% and −7.19%±24.24%, respectively, resulting in significant between- group differences of the right side (Fig. 3B). In contrast, Dm of the left and right VL decreased in the CON group by −7.74%± 26.33% and −7.25%±23.83%, respectively, but increased in the FH group by 9.74%±21.02% and 7.89%±25.39%, respectively, producing significant between-group differences (Fig. 3C and D). Likewise, Dm of the left and right BF decreased in the CON group by −14.99%±17.38% and −17.76%±17.23%, respectively, while it increased in the FH group by 15.89%±24.23% and 17.21%±25.08%, respectively, again indicating significant between-group differences (Fig. 3E and F).
Differences and changes in erythrocytes factors
As shown in Table 3, baseline levels of erythrocytes subtypes did not differ significantly between the CON and FH groups. After the intervention, no significant interaction effects between groups were observed for Hb, MCH, and RDW, whereas significant group-by-time interaction effects were found for HCT, MCV, and MCHC.
Fig. 4 presents the Δ% changes in erythrocyte indices between the two groups from pre- to postintervention. HCT showed an increase of 0.39%±3.48% in the CON group, whereas it decreased by -1.94%±4.59% in the FH group, demonstrating significant group differences (Fig. 4A). MCV rose by 1.67%±0.89% in the CON group and by 1.02%±1.30% in the FH group, also indicating significant between-group differences (Fig. 4B). MCHC declined in both groups, with a reduction of −1.26%±1.16% in the CON group and −0.35%±1.07% in the FH group, again showing significant differences between groups (Fig. 4C).
Differences and changes in leukocytes’ subtypes
As shown in Table 4, baseline levels of leukocytes’ subtypes did not differ significantly between the CON and FH groups. Following the intervention, the NK cells to CD8+ T cells decreased in the CON group but increased in the FH group, demonstrating significant interaction effects.
Fig. 5 illustrates the differences in lymphocyte subtypes between the two groups, expressed as the Δ% change from pre- to postintervention. NK cells decreased in the CON group by −2.60%± 38.23% but increased in the FH group by 44.95%±41.88%, resulting in significant between-group differences (Fig. 5A). NKT cells decreased in the CON group by −14.17%±30.98% but increased in the FH group by 22.92%±37.36%, also showing significant between-group differences (Fig. 5B). CD19+ B cells decreased in the CON group by −14.51%±15.16% but increased in the FH group by 14.81%±23.52%, yielding significant between- group differences (Fig. 5C). CD3+ T cells decreased in the CON group by −9.97%±20.34% but increased in the FH group by 10.60%±19.52%, showing significant between-group differences (Fig. 5D). CD4+ T cells decreased in the CON group by −13.93%± 19.76% but increased in the FH group by 13.91%±22.41%, demonstrating significant between-group differences (Fig. 5E). CD8+ T cells decreased in the CON group by −11.93%±19.56% but increased in the FH group by 21.56%±45.25%, also resulting in significant between-group differences (Fig. 5F).
DISCUSSION
This study aimed to investigate intramuscular oxygen utilization in older adults during FH, along with associated hematological and immunological variables. The findings revealed that SmO2 levels in the FH group progressively declined during a 120-min hike, reaching their lowest point between 60 and 90 min. After engaging in this activity twice weekly for 4 weeks, the FH group demonstrated significant improvements in Tc of the VL, as well as enhanced Dm in both the VL and BF compared to the CON group. In addition, favorable changes were observed in erythrocyte-related parameters, including HCT, MCV, and MCHC. Furthermore, immune cell subsets increased toward the higher end of their normal ranges in the FH group, indicating a potential enhancement in immune function.
Skeletal muscle aging is a complex process characterized by a decline in muscle mass, which in turn leads to reductions in muscle strength and function (Nilwik et al., 2013; Wilkinson et al., 2018). The loss of muscle mass occurs partially through muscle fiber atrophy and motor unit loss, but even these partial changes can result in decreased mobility, increased risk of falls, disability, loss of independence, and a reduced quality of life (Shafrin et al., 2017). Therefore, understanding the factors that contribute to muscle aging is crucial for maintaining optimal muscle function and promoting healthy aging. Recent findings from TMG have reported that power-trained athletes exhibit shorter Tc in the BF, while individuals with pathological conditions show shorter Tc in the VL and gastrocnemius medialis. In contrast, endurance-trained athletes display longer Tc values across all three muscles (Pus et al., 2023). A shorter Tc in the VL has been shown to correlate with a lower proportion of slow-twitch (ST) myosin heavy chain fiber (Simunič et al., 2011), supporting earlier findings (Dahmane et al., 2001). Collectively, these results suggest that anaerobic exercise may promote fast-twitch (FT) fiber characteristics during aging. Aging induces a shift from ST to FT muscle fibers, with FT fibers being more susceptible to early energy depletion and fatigue. Previous studies indicate that endurance-trained athletes possess a higher proportion of ST and FT-IIa fibers in the VL compared to power-trained athletes and nonathletes (Pollock et al., 2018). Consequently, longer Tc would be expected across multiple muscles, irrespective of sex or age. However, although hiking in mountainous terrain is generally classified as an aerobic endurance activity, the fact that the participants were older adults suggests that the repeated uphill and downhill walking may have required greater recruitment of FT II fibers rather than ST fibers. Furthermore, after performing mountain hiking, the observed reduction in Tc among older adults indicates that the anaerobic demands of hiking may promote FT fiber characteristics during aging. Additionally, shorter Tc has been correlated with a lower proportion of ST myosin heavy chain fibers in the VL, supporting prior findings on fiber-type distribution and contraction dynamics (Dahmane et al., 2001; Simunič et al., 2011). Collectively, these results underscore the complex interplay between aging, exercise modality, and muscle fibers’ composition in determining muscle function and highlight the potential for targeted interventions in older adults. Notably, the lowest Dm in nursing home residents indicates that immobility is associated with reduced Dm (Fabiani et al., 2021). This decline may indicate changes associated with aging and inactivity, such as diminished contractile efficiency resulting from impaired neuromuscular junction activity and fibrosis caused by excessive scar tissue accumulation (Tieland et al., 2018), or a loss of muscle fiber elasticity, possibly linked to selective atrophy and a shift toward ST fiber remodeling (Ochala et al., 2007). These findings highlight the potential value of interventions, such as forest hiking, to preserve or enhance muscle contractile properties, particularly in postural muscles, and to mitigate the adverse effects of aging and immobility on muscle function.
Recent research has shown that spending time in forest environments can help reduce stress and promote relaxation (Li, 2022). Beyond the potential for improving muscle function mentioned above, walking or hiking in forests may offer a variety of additional health benefits for older adults. In general, increases in Hb levels are observed during moderate- to high-intensity exercise, as such activities place greater demands on the oxygen transport capacity, leading to compensatory elevations postexercise. Previous studies have generally reported that exercise training moderately increases HCT in young and middle-aged adults, whereas similar improvements are not observed in older adults. These findings suggest that adaptations of HCT, plasma volume, and total blood volume in response to exercise may differ by age. However, there is still no consensus regarding the effects of exercise training on HCT, as inconsistent results have been reported across studies (Bonne et al., 2014; Gass et al., 2004; Helgerud et al., 2007; Okazaki et al., 2009). In the present study, erythrocytes and Hb showed slight decreases in both the FH and CON groups, with no significant between-group differences. Notably, the FH group demonstrated a slight decline in HCT and MCHC, alongside a slight increase in MCV, resulting in a significant interaction effect between groups. These results suggest that the forest hiking may have corresponded to moderate-to-high exercise intensity for the participants. This interpretation is further supported by the observation that SmO2 levels dropped to nearly below 40% between 60 and 90 min of hiking, while heart rate values approached 80% of VO2max during the same period, indicating the strenuous nature of the activity. In this context, McLaren et al. (1987) reported that both short- and long-term exercise training led to a modest and consistent increase of approximately 81 mL in HCT among healthy young and middle-aged individuals. Although various mechanisms have been proposed to explain exercise-induced erythropoiesis (Hu and Lin, 2012), these findings support the hypothesis that exercise training provides relatively limited stimulation for HCT adaptation in older adults (Schmidt and Prommer, 2010). Meanwhile, compared with sedentary individuals, physically trained individuals have been reported to exhibit higher Hb but lower HCT levels (Brun et al., 2000). In older adults, physical exercise can increase erythrocytes and thereby enhance the oxygen-carrying capacity of the blood, and the beneficial effects of endurance training on erythrocytes are well established regardless of age (Szygula, 1990). However, a study analyzing hematological parameters after a 6-month endurance training program in older individuals reported no significant changes in erythrocytes, thrombocytes, leucocytes, Hb, HCT, MCV, MCH, or MCHC (Bobeuf et al., 2009). These findings highlight the need for further research to clarify the effects of exercise on erythrocyte-related variables.
During exercise, leukocytes are mobilized into the peripheral blood, leading to increased concentrations of neutrophils, lymphocytes, and monocytes. The rise in lymphocyte levels is thought to reflect the mobilization of all lymphocyte subsets, including NK cells, T cells, and B cells (Rooney et al., 2018). However, the effects of exercise vary depending on intensity: unlike moderate exercise, strenuous exercise has been associated with reductions in lymphocyte concentrations and impairments in cell-mediated immunity (Hoffman-Goetz and Pedersen, 1994). Woods et al. (1998) investigated the effects of 6 months of moderate aerobic exercise on T-lymphocyte and NK-cell function in sedentary older adults (65±0.8 years). Participants were randomly assigned to an exercise intervention group or a CON group. Following the 6-month intervention, no significant changes were observed in leukocyte, neutrophil, lymphocyte, monocyte, eosinophil, or basophil counts. Additionally, the percentages of CD3+, CD4+, and CD8+ T cells remained unchanged. Meanwhile, Ceddia et al. (1999) investigated leukocyte recruitment and T-lymphocyte function in 33 older adults (65.3±0.8 years) and 14 young controls (22.4±0.7 years) in response to a bout of maximal exercise. Both age groups exhibited exercise-induced leukocytosis; however, the magnitude of the response was lower in the older group. Immediate postexercise samples showed similar increases in CD3+ cell numbers in both groups, but older adults recruited fewer CD4+ cells and more CD8+ cells, although the significance of this difference was unclear.
Across ages, approximately equal proportions of memory and naïve CD8+ cells were recruited, whereas exercise induced a significant increase in CD4+ memory cells and a concomitant decrease in CD4+ naïve cells in older adults compared to young subjects. Similarly, Rincón et al. (1996) investigated the effects of exercise training on NK-cell activity in frail elderly men aged 70 years or older. Following a 3-month exercise program conducted 3 times per week, NK-cell cytotoxicity significantly decreased compared with control participants who did not undergo the intervention. In this study, forest hiking led to increases in both leukocyte and lymphocyte subsets. These findings contrast with those of Woods et al. (1998) but are consistent with the results reported by Ceddia et al. (1999) and Rincón et al. (1996). Notably, differentiated lymphocyte populations, including NK cells, CD3+ cells, CD4+ cells, and CD8+ cells, were elevated, suggesting that forest hiking may enhance immune function in older adults. Nevertheless, older adults can maintain the capacity to mobilize T lymphocytes and NK cells through exercise, and the observed increase in CD19+ B cells indicates that forest hiking can have a meaningful positive impact on immune function in the elderly. The recruited T lymphocytes are predominantly previously activated cells with a higher replicative history compared with resting cells. It remains unclear whether this recruitment differentially affects immune cell function in young versus older adults. Importantly, exercise-induced increases in NK-cell cytotoxicity are preserved in older adults, and training may enhance this capacity to elevate NK cytotoxicity in response to exercise (Bruunsgaard and Pedersen, 2000). However, highly conditioned older adults seem to exhibit relatively better-preserved immune function, although it remains unclear whether this is attributable to exercise training itself or to other lifestyle-related factors.
Despite these findings, this study has several limitations. First, although the sample size was determined using scientific methods, the participants may not fully represent the wider older adult population. Second, as all participants were of East Asian descent, the applicability of these interventions to older individuals from other ethnic backgrounds may be limited. Third, while this study focused on hematological and immunological factors, it is important to acknowledge that the scientific community recognizes a broader range of relevant biomarkers beyond those examined here. Therefore, further research involving larger and more diverse cohorts, as well as assessments of a wider array of blood biomarkers, is warranted to better understand the effects of exercise combined with forest bathing.
Eventually, this study investigated intramuscular oxygen utilization during forest hiking and found that SmO2 levels gradually decreased over the total 120 min of hiking, reaching their lowest point between 60 and 90 min. Furthermore, engaging in forest hiking twice a week for 4 weeks led to positive changes in muscle function, influenced HCT and Hb, and increased immune cell subtypes to upper-normal levels.
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