Pro-Inflammatory and Anti-Inflammatory Cytokines in Older Men Participating in an Aquatic Exercise Programme and a Multicomponent Exercise Programme: A Cross-Sectional Exploratory Study

1. Introduction

Ageing is accompanied by a range of physiological alterations, including changes in immune system function. Among these, immunosenescence, defined as the progressive deterioration of immune competence, and inflammaging, characterised by a state of chronic low-grade systemic inflammation, represent two hallmark features of the ageing process [1]. These age-related immune alterations have been associated with an increased susceptibility to chronic diseases, impaired responses to physiological and environmental stressors, and a greater risk of functional decline in older adults [2].

Cytokines are low-molecular-weight signalling proteins that play a pivotal role in regulating immune and inflammatory responses. Pro-inflammatory cytokines, such as interleukin (IL)-1β and tumour necrosis factor-alpha (TNF-α), initiate and amplify inflammatory processes, whereas anti-inflammatory cytokines, including IL-10 and IL-1 receptor antagonist (IL-1ra), counterbalance these responses by limiting excessive inflammation and promoting immune homeostasis [3]. With advancing age, the balance between pro- and anti-inflammatory mediators may become dysregulated, contributing to the development and maintenance of chronic low-grade inflammation, a phenomenon referred to as inflammaging [1,4]. Consequently, the combined assessment of pro- and anti-inflammatory cytokines provides valuable insight into the inflammatory profile of older adults and may contribute to a better understanding of the potential immunomodulatory effects of regular physical exercise.

Regular physical exercise has been recognised as an important non-pharmacological strategy for mitigating some of the age-related alterations in immune function. Recent evidence suggests that exercise may modulate both pro- and anti-inflammatory cytokine profiles; however, findings remain inconsistent owing to differences in exercise modality, programme duration, and the characteristics of the study populations [1,5].

Among the exercise modalities most adopted by older adults, aquatic exercise and multicomponent exercise programmes are particularly noteworthy because they are accessible, are frequently delivered in a group-based setting, and can be tailored to the functional capacities of participants. Furthermore, recent evidence from the Portuguese population indicates that group-based exercise classes and aquatic activities are among the most frequently practised forms of physical exercise, highlighting the relevance of these modalities in promoting active ageing [6].

Current international guidelines for older adults advocate exercise programmes incorporating aerobic, muscle-strengthening, balance, and flexibility components, all of which are key features commonly integrated into these exercise modalities [7]. Recent studies have also demonstrated that both aquatic exercise and multicomponent exercise programmes improve mobility, muscle strength, and overall physical fitness in older adults [8,9]. However, evidence regarding their effects on inflammatory biomarkers remains limited.

Despite the growing body of research in this field, the available evidence remains constrained by two important limitations. First, studies involving exclusively male samples are scarce, as participants in both aquatic and multicomponent exercise interventions are predominantly women. For instance, studies investigating aquatic exercise programmes in older adults have consistently reported a marked predominance of female participants [10,11], a pattern that is also evident in studies examining multicomponent exercise programmes in older adults [6,9]. Second, there is a lack of direct comparisons between these exercise modalities with respect to pro- and anti-inflammatory cytokine profiles. Therefore, further research is warranted to investigate these exercise modalities in older men and to compare two of the most practised forms of exercise in the context of active ageing concerning inflammatory biomarkers.

The present study aimed to compare the profiles of pro- and anti-inflammatory cytokines, used as measurable biochemical biomarkers of the inflammatory response associated with exercise, in older men participating in an aquatic exercise programme (AE) and a land-based multicomponent exercise programme (ME). Given the cross-sectional design of the study, the primary objective was to explore whether statistically significant differences existed in the levels of pro- and anti-inflammatory cytokines between older men engaged in these two exercise modalities. This approach was based on the fact that both programmes shared broadly comparable characteristics in terms of exercise frequency and intensity, differing primarily in the environment in which they were delivered (aquatic versus land-based). Although aquatic exercise may confer specific benefits for older adults, it remains unclear whether these potential advantages are reflected in the cytokine profiles examined in the present study.

2. Materials and Methods

2.1. Study Design

A cross-sectional exploratory study was conducted involving 15 older men, allocated to two groups according to the exercise modality practised: aquatic exercise (AE; n = 7) and multicomponent exercise (ME; n = 8). The primary outcomes comprised the assessment of the cytokines IL-1β, TNF-α, IL-10, and IL-1 receptor antagonist (IL-1ra). Secondary variables included body composition and participant characteristics obtained through questionnaires addressing medical history and lifestyle factors. All variables were assessed at a single time point.

The study was conducted in accordance with the ethical principles of the Declaration of Helsinki and was approved by the Ethics Committee of the Faculty of Sport Sciences and Physical Education, University of Coimbra (reference: CE/FCDEF-UC/00462019). All participants received detailed information regarding the study objectives and procedures and provided written informed consent prior to data collection. Participant anonymity, data confidentiality, and the right to withdraw from the study at any time without penalty were ensured.

Blood samples were coded immediately following collection. Biological sample processing was performed by a laboratory technician blinded to group allocation. Statistical analyses were conducted using a coded dataset, with group identities concealed throughout the analytical process.

2.2. Participants

Participants were recruited from the district of Castelo Branco, Portugal, according to the following inclusion criteria: male sex; age ≥ 60 years; community-dwelling status; regular participation in supervised aquatic exercise or multicomponent exercise programmes for at least 6 months; and willingness to participate, confirmed by providing written informed consent. The exclusion criteria were attendance below 75% of the scheduled exercise sessions during the preceding 6 months and inability to complete the assessment procedures required for the study.

2.3. Exercise Programmes

Participants were enrolled in one of two regular exercise programmes, which differed in terms of the exercise environment and the predominant characteristics of the intervention. The AE group comprised participants engaged in an aqua aerobics programme conducted in a heated indoor swimming pool, with a predominantly aerobic focus. Sessions included dynamic locomotor exercises, choreographed movement sequences, and aqua aerobics exercises performed with and without aquatic equipment, all adapted to the participants’ functional capacities.

The multicomponent exercise (ME) group comprised participants enrolled in a land-based senior fitness programme. Sessions incorporated multiple components of functional fitness, including joint mobility, muscle-strengthening, balance, coordination, flexibility, and aerobic endurance exercises.

In both groups, participants had attended their respective exercise programme for at least 6 months (mean attendance rate: 85.49%). All sessions were supervised by qualified exercise and sport science professionals and delivered in mixed-sex groups. Exercise sessions lasted 45 minutes and were conducted twice weekly. Exercise intensity was maintained at a moderate level and monitored using the Borg Category-Ratio 10 (CR10) Rating of Perceived Exertion scale [12].

2.4. Instruments

2.4.1. Participant Characterisation Questionnaire

At baseline, all participants completed a questionnaire developed by the research team to characterise the study sample. The questionnaire collected information on marital status, infections, allergies, medical conditions, average daily sleep duration, sleep quality, and the consumption of alcohol, tobacco, and recreational drugs.

2.4.2. Body Composition

All anthropometric measurements were performed by the same investigator, who had received prior training in the assessment procedures. The following parameters were evaluated: (i) height, measured using a portable stadiometer (Seca Bodymeter^®, model 208, Hamburg, Germany; precision 0.1 cm); (ii) body mass, body mass index (BMI), visceral fat, fat mass (FM), and muscle mass (MM), assessed using a portable scale (TANITA^® BC-601).

2.4.3. Cytokines

Venous blood samples were collected after a 12-hour overnight fast. A total of 15 mL of blood was obtained from each participant and processed under standard laboratory conditions. Blood samples were aliquoted into three collection tubes, centrifuged at 3,500 rpm for 10 minutes, and the resulting serum was transferred to cryogenic storage tubes. Serum concentrations of IL-1β, IL-1 receptor antagonist (IL-1ra), IL-10, and TNF-α were subsequently quantified using enzyme-linked immunosorbent assay (ELISA) kits (Invitrogen^®, CA, USA; Bender MedSystems GmbH, Vienna, Austria). Cytokine concentrations were expressed in picograms per millilitre (pg/mL).

2.5. Data Collection Procedures

Data collection was conducted in three consecutive stages. During the first visit to the facilities where the exercise sessions took place, the study was presented to the participants, and written informed consent was obtained. During the second stage, which took place at the subsequent exercise session, participants completed the characterisation questionnaire, and body composition assessments were performed. These assessments were conducted individually in a dedicated, temperature-controlled room to ensure participant privacy. All participants were assessed under standardised conditions by the same research team.

During the third stage, participants attended a local clinical laboratory, where venous blood samples were collected following a 12-hour overnight fast. All blood samples were obtained during the morning to standardise assessment conditions and minimise potential circadian variation in cytokine concentrations. Blood collection was performed by a certified phlebotomist, after which the samples were transported to the laboratory under appropriate storage and handling conditions for subsequent processing and analysis.

2.6. Statistical Analysis

Given the exploratory nature of the study and the limited availability of eligible participants, no a priori sample size calculation was performed. A post hoc sensitivity analysis was subsequently conducted using G*Power [13], assuming two independent groups (AE: n = 7; ME: n = 8), a significance level of α = 0.05, and statistical power of 80%. The analysis indicated that the study design was only capable of detecting very large effect sizes (approximately Cohen’s d = 1.57). Consequently, the findings should be interpreted with caution, emphasizing effect sizes and confidence intervals, rather than solely on p-values.

Descriptive statistics were calculated for all study variables. Normally distributed variables are presented as mean, standard deviation (SD), minimum, and maximum values, whereas non-normally distributed variables are reported as median and interquartile range (IQR; 25th–75th percentile). Data normality was assessed using the Shapiro–Wilk test, kurtosis, and visual inspection of histograms, while homogeneity of variances was evaluated using Levene’s test. Between-group comparisons were performed using the independent-samples Student’s t-test when assumptions were met, Welch’s t-test when homogeneity of variances was violated, and the Mann–Whitney U test for non-normally distributed variables. In addition, 95% confidence intervals (CIs) for the mean difference were reported. Effect sizes were quantified using Hedges’ g for parametric analyses and the rank-biserial correlation for the Mann–Whitney U test. Statistical analyses were performed using Jamovi software (version 2.6.45), with statistical significance set at p < 0.05.

3. Results

Regarding the sample characteristics (Table 1), the study included 15 older men allocated to two groups: AE n = 7; 70,8 ± 4,5 years and ME n = 8; 75,4 ± 4,1 years. Participants in the AE group were younger than those in the ME group. This difference reached statistical significance (p = 0,050) and was associated with a large effect size, suggesting a potential age imbalance between the groups.

Relative to marital status, all participants were married except for one participant, who was widowed. Regarding regular medication use, all participants in the ME group and six participants in the AE group reported taking prescribed medication regularly. Concerning medical history, three participants reported previous urinary tract infections; however, none reported an active infection or symptoms consistent with infection at the time of blood collection. Reported allergies included pollen and bee venom allergies. Self-reported medical conditions included hypertension, diabetes mellitus, and hypercholesterolaemia in both groups, while obstructive sleep apnoea was additionally reported in the AE group.

Mean sleep duration was 7,29 hours per night in the AE group and 7,25 hours per night in the ME group. Regarding sleep quality, 12 participants rated their sleep as good and three as satisfactory. None of the participants reported current alcohol consumption, tobacco use, or recreational drug use.

Concerning body composition, the AE group presented lower mean values for body mass and fat mass percentage (FM%), similar mean values for visceral fat, and higher mean values for muscle mass percentage (MM%) than the ME group. Following verification of the assumptions of normality and homogeneity of variances, the independent-samples Student’s t-test was applied to all variables except FM%, for which Welch’s t-test was used owing to violation of the homogeneity of variances assumption. No statistically significant between-group differences were observed for any of the body composition variables analysed. Effect sizes were small for height, body mass, visceral fat, and FM%, and moderate for MM%. However, the observed difference in muscle mass percentage should be interpreted with caution, given the exploratory nature of the study and the small sample size (Table 2).

About the cytokines assessed, the pro-inflammatory cytokines IL-1β and TNF-α showed lower median concentrations in the AE group than in the ME group. Among the anti-inflammatory cytokines, IL-10 exhibited very similar median concentrations in both groups, whereas IL-1 receptor antagonist (IL-1ra) showed a higher median concentration in the ME group. As the assumption of normality was not met for the cytokine variables, between-group comparisons were performed using the Mann–Whitney U test for independent samples. No statistically significant between-group differences were observed for any of the cytokines analysed, and effect sizes ranged from trivial to small (Table 3).

4. Discussion

The present study aimed to compare pro- and anti-inflammatory cytokine profiles in older men participating in an aquatic exercise programme and a land-based multicomponent exercise programme. The main findings showed no statistically significant differences between the two groups in the cytokines analysed, namely IL-1β, TNF-α, IL-10, and IL-1ra. Likewise, no significant differences were observed in the body composition variables used to characterise the study sample. These findings suggest that, within this sample, both exercise modalities were associated with comparable inflammatory profiles.

The exploratory nature of the present study should be considered when interpreting the absence of statistically significant differences between groups. Nevertheless, these findings are of interest, as they provide a direct comparison between two exercise modalities that are widely practised by older adults but have rarely been investigated in exclusively male populations.

Regular physical exercise may contribute to the modulation of the inflammatory profile in older adults, particularly through reductions in pro-inflammatory markers such as TNF-α, IL-6, and C-reactive protein [14]. However, the effects of exercise on anti-inflammatory cytokines, including IL-10, appear to be less consistent and may depend on factors such as the duration, intensity, and type of exercise programme performed [1]

In the context of aquatic exercise [15], a randomised controlled trial involving community-dwelling older adults reported that different aquatic exercise programmes performed over a 28-week period improved the systemic inflammatory profile, with participants exhibiting a shift towards a less pro-inflammatory state, whereas the non-exercising control group showed the opposite pattern. In the same study, TNF-α concentrations decreased in all exercise groups (aerobic, interval, and combined), while IL-10 concentrations increased in the interval training group. These findings suggest that aquatic exercise may play an important role in attenuating the chronic low-grade inflammation associated with ageing. In the present study, although the aquatic exercise group exhibited lower median concentrations of TNF-α and IL-1β than the multicomponent exercise group, these differences did not reach statistical significance.

Regarding multicomponent exercise, previous studies have also reported favourable effects on inflammatory and functional outcomes in older adults. In a randomised controlled trial involving frail older adults, [16] found that a multicomponent exercise programme incorporating aerobic, resistance, and balance training reduced inflammatory markers, including IL-6 and C-reactive protein, while also improving measures of physical performance. Similarly, [14] reported that combined aerobic and resistance exercise programmes in older adults reduced TNF-α and IL-6 concentrations and increased IL-10 concentrations, suggesting an improvement in immune regulation. Collectively, these findings indicate that well-structured and supervised land-based exercise programmes may also contribute to a more favourable inflammatory profile.

Despite these findings, direct comparisons between aquatic and land-based exercise with respect to cytokine profiles remain limited. Recent systematic reviews comparing aquatic and land-based exercise interventions in older adults have primarily focused on outcomes such as physical fitness, body composition, and quality of life, with considerably less attention given to inflammatory biomarkers [17]. The present study adds preliminary evidence by comparing two widely practised exercise modalities in older men, suggesting that both are associated with similar inflammatory profiles when performed regularly under comparable conditions of exercise frequency, duration, and intensity. Accordingly, the findings do not indicate the superiority of either exercise modality over the other with respect to the inflammatory biomarkers assessed.

This study has several limitations that should be acknowledged. First, the small sample size limited the statistical power of the analyses. Second, the cross-sectional design precludes the establishment of causal relationships between participation in the exercise programmes and the observed inflammatory profiles. Third, the absence of a sedentary control group prevented the assessment of whether both exercise groups exhibited a more favourable inflammatory profile than older adults who did not engage in regular exercise. Fourth, the use of a single blood sampling time point may not have captured the natural variability of circulating cytokine concentrations, as these biomarkers are influenced by factors such as recent infections, medication use, sleep, dietary intake, and overall clinical status. Finally, a significant age difference was observed between the groups, with participants in the ME group being older, on average, than those in the AE group. Given that inflammaging is intrinsically associated with the ageing process, this age imbalance may have acted as a potential confounding factor in the comparison of cytokine profiles between the two exercise modalities.

Future research should further investigate this comparison using longitudinal and intervention study designs, ideally including a sedentary control group. It will also be important to recruit larger samples with a more balanced representation of men and women, control for potential confounding factors such as medication use, chronic diseases, dietary intake, and sleep, and evaluate a broader panel of inflammatory biomarkers. Furthermore, incorporating multiple assessment time points would provide a better understanding of whether changes in cytokine profiles are attributable to regular physical exercise or reflect pre-existing participant characteristics.

Notwithstanding these limitations, the present study highlights the importance of comparative research in this population by providing preliminary evidence on the potential role of different exercise settings in modulating the inflammatory response associated with ageing.

5. Conclusions

The findings of the present study suggest that older men participating in aquatic exercise and land-based multicomponent exercise programmes exhibit similar inflammatory profiles with respect to the cytokines assessed. No statistically significant between-group differences were observed in the pro-inflammatory markers IL-1β and TNF-α or in the anti-inflammatory markers IL-10 and IL-1 receptor antagonist (IL-1ra). These findings indicate that, within this sample, both exercise modalities may be associated with comparable inflammatory responses. However, given the cross-sectional design and the small sample size, further studies involving larger cohorts, longitudinal designs, and controlled intervention trials are required to clarify the effects of these exercise modalities on the inflammatory profile of older men.