Layout 1 In the past decade, the understanding of skeletal muscle as a secretory organ has significantly increased. Skeletal muscle synthesizes and secretes into the bloodstream a wide range of myokines which exerts beneficial effects on various tissues and organs in an autocrine, paracrine, and endocrine manner. Among them there is irisin, mainly produced in response to muscle contraction. Indeed, physical exercise (PE) activates the Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) which determines the increase of Fibronectin type III domain-containing protein 5 (FNDC5) expression.1 Irisin is derived from the proteolytic cleavage of the extra cytoplasmatic region of FNDC5 protein and it is consequently released into the peripheral circulation.1 This myokine may be considered one of main mediators of the beneficial effects of PE on human health, given that it promotes cognition and neurodevelopment by triggering the expression of Brain-Derived Neurotrophic Factor (BDNF) and plays key functions in the whole-body metabolism. It induces the browning of white adipocytes, by increasing the expression of mitochondrial Uncoupling protein 1 (UCP1), improving thermogenesis and weight loss.2 Moreover, it helps to maintain bone homeostasis,3 modulates metabolic processes in the liver,4 enhances glucose metabolism and promotes muscle hypertrophy in human muscle cells.5,6 A considerable amount of literature has been published on irisin response to an acute bout of exercise in a period ranging from zero minutes to two h after the physical sti- mulus.7,8 However, the comparison between studies is complicated due to various type, intensity, and duration Abstract This study aimed to analyze the acute impact of exercise on serum irisin levels in 22 young (YA, 24.6±3.5 yrs) and in 12 middle-aged male adults (MA, 54.6±5.7 yrs) 15 min and 24 h after an incremental cycling exercise test to exhaustion. ELISA assay was used for serum irisin detection. Circulating irisin increased significantly from baseline (9.0±2.0 ng/ml) to 15 min post-exercise (10.2±2.0 ng/ml, P <0.001), but the greatest increment was detected after 24 h (13.5±2.5 ng/ml, P <0.001) reaching more than 50% of the basal release. Levels were significantly higher in YA (9.7±1.7 to 11.1±1.8 to 14.5±2.2 ng/ml) than MA (7.6±1.6 to 8.7±1.5 to 11.8±2.2 ng/ml) for all measured time-points (P <0.05). Nevertheless, MA showed a comparable increase in serum irisin levels when compared to YA. These findings highlight the importance of acute physical exercise as a countermeasure against age-related deterioration of skeletal muscle mass and function in both YA and MA. Key Words: acute physical exercise; irisin; myokine; serum; aging. Eur J Transl Myol 34 (2) 12693, 2024 doi: 10.4081/ejtm.2024.12693 - 177 - Thematic Section: Advances in Musculoskeletal and Neuromuscular Rehabilitation | Maccarone & Masiero Eur J Transl Myol 34 (2) 12693, 2024 doi: 10.4081/ejtm.2024.12693 The time course of irisin release after an acute exercise: relevant implications for health and future experimental designs Ester Tommasini,1,2,3 Sara Missaglia,1,2 Paola Vago,1,2 Christel Galvani,2,3 Claudio Pecci,4 Ermanno Rampinini,4,5 Andrea Bosio,4 Andrea Morelli,4 Andrea Bonanomi,6 Andrea Silvestrini,7 Alvaro Mordente,7 Daniela Tavian1,2 1Laboratory of Cellular Biochemistry and Molecular Biology, CRIBENS, Università Cattolica del Sacro Cuore, Milan, Italy; 2Department of Psychology, Università Cattolica del Sacro Cuore, Milan, Italy; 3Exercise & Sport Science Laboratory, Università Cattolica del Sacro Cuore, Milan, Italy; 4Human Performance Laboratory, MAPEI Sport Research Centre, Olgiate Olona (VA), Italy; 5Sport and Exercise Discipline Group, Human Performance Research Centre, Faculty of Health, University of Technology Sydney, Moore Park, NSW, Australia; 6Department of Statistical Science, Università Cattolica del Sacro Cuore, Milan, Italy; 7Department of Basic Biotechnological Sciences, Intensivological and Perioperative Clinics, Università Cattolica del Sacro Cuore, Rome, Italy. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Non -co mmerc ial us e o nly Irisin release after an acute exercise Eur J Transl Myol 34 (2) 12693, 2024 doi: 10.4081/ejtm.2024.12693 of exercise protocols, as well as heterogeneous study population enrolled. Furthermore, the timing of blood drawing is highly variable after exercise.7 There seem to be a dose-response relationship between exercise and iri- sin elevation,9,10 but contradictory results have been re- ported considering research that evaluate the irisin response to an incremental exercise until exhaustion. In particular, acute responses immediately (0-15 minutes) after a maximal incremental exercise test have shown either a slight rise9,11-13 or no effects14 on serum or plasma irisin levels, while the studies that have measured the amount of circulating irisin at least 30 min following an incremental exercise to exhaustion did not find signifi- cant changes in the myokine concentration.12,13 The ex- isting research seem to suggest that the elevation of irisin following an incremental exercise test to exhaustion is transient, and it is likely that after 24 hours the levels have returned to baseline conditions. However, no studies reported the time course of serum irisin from baseline, soon after as well as 24 hours post the physical exercise. Given that muscle mass is the main predictor of the cir- culating irisin concentration in human,15 the age-related decay in skeletal muscle mass would suggest a reduction in circulating irisin concentration.16 The relationship be- tween irisin and age has already been discussed by dif- ferent authors,11,17 while few studies have examined the impact of acute bout of exercise on irisin release by com- paring different age groups, revealing contradictory re- sults.18-21 In fact, Huh et al.18 have demonstrated that plasma irisin increases immediately after 45-min of vig- orous-to-exhaustive exercise and the increment was sim- ilar in both young and older adults. On the contrary, others have reported no significant changes in plasma or serum irisin levels in response to either a single bout of circuit training in both young and middle-aged/older groups of females19 and males,20 as well as after 180 mi- nutes of moderate-intensity treadmill walking in both young and older adult groups.21 Currently, no previous study has explored the irisin mod- ulation from baseline, immediately and one day after an incremental exercise to exhaustion in a substantial number of healthy male subjects. In addition, there are limited and conflicting information regarding the age-re- lated differences in irisin secretion after an exogenous stimulus. The aims of the present research were to eval- uate the impact of a single bout of incremental exercise on a 24 hours’ time course of serum irisin levels in healthy male subjects and to compare the acute exercise- induced irisin changes in young and middle-aged adults, thus enhancing the comprehension of the exercise’s in- fluence on this myokine. We hypothesized that an acute stimulus could induce an immediate increase in irisin levels, as well as several hours after exercise. Given the age-related decline in skeletal muscle mass, we also hypothesized that the acute exercise-induced changes in irisin levels would differ between young and middle-aged adults, with younger adults showing a more pronounced increase compared to their older counterparts. Material and Methods Participants Thirty-four healthy males (aged 20-65 yrs) were recruited via notices (recruitment start date: June 8, 2022; recruitment end date: December 12, 2022). Data were collected and an- alyzed from June to December 2022. The participants were assigned either to the young adult group (YA) (n=22, 24.6±3.5 yrs) or middle-aged adult group (MA) (n=12, 54.6±5.7 yrs). The cutoff point used correspond to 40 yrs and is reasonable for dividing the two groups.22 This study was conducted in accordance with the ethical principles es- tablished in the Declaration of Helsinki. The protocol of the present study was approved by the Ethics Commission of the Università Cattolica del Sacro Cuore of Milan (N° of protocol 28-22) and written informed consent for study par- ticipation, permission for personal data treatment and bio- chemical analysis was obtained from all participants upon enrolment. Individuals with a body mass index (BMI) ≤30 kg/m2 were involved in the study. To ensure the general health of the subjects, a self-reported history of neurological disorders, musculoskeletal impairment, other motor restric- tions which could influence the regular outcome of the study, and current pharmacotherapy, which could alter the results, were set as the exclusion criteria. Participants com- pleted all the assessments in two consecutive days and were instructed to eat a light breakfast and avoid smoke, alco- holic and caffeinated products at least 2 h before the test. Moreover, they were asked to refrain from moderate and vigorous intensity, and long-duration training in the pre- vious 48 h and for the duration of the study post-exercise. Assessments took place at the same time of the day, from 10:00 a.m. to 1:00 p.m., taking into consideration the cir- cadian effects. Anthropometric and body composition parameters The analysis of anthropometric and body composition pa- rameters included weight and height measurement, the cal- culation of BMI, the estimation of body density and percentage of fat mass (FM). weight and height were meas- ured using a mechanical scale (761, SECA GmbH & Co. KG., Hamburg, Germany) and a stadiometer (213, SECA GmbH & Co. KG. Hamburg, Germany), respectively. Per- centage of FM was estimated by skinfold thickness using a calibrated skinfold caliper (Harpenden; Baty International, United Kingdom), in accordance with the procedures sug- gested by the American College of Sports Medicine.23 Physical activity Short form of the Italian version of International Physical Activity Questionnaire was used to evaluate the volume of physical activity over the previous 7 days.24 Total physical activity (PAtot) was calculated by summing the time spent on walking, moderate, and vigorous intensity activities and was expressed in metabolic equivalent of task (MET)-mi- nutes per week. Exercise protocol All the participants performed a maximal incremental test on a cycle ergometer. The exercise test took place in a room - 178 - Non -co mmerc ial us e o nly Irisin release after an acute exercise Eur J Transl Myol 34 (2) 12693, 2024 doi: 10.4081/ejtm.2024.12693 with a relative humidity less than 60% and a temperature ranging from 18°C to 22°C. After 2 min of basal oxygen uptake assessment on the cycle ergometer (LC6 Monark; Vansbro, Sweden and Excalibur Sport, Lode BV, Gron- ingen, Netherlands), the test started at 80 W and, the load was increased by 20 W every minute until volitional ex- haustion. The subjects were instructed to maintain a ca- dence between 85 and 90 revolutions per minute. The exercise protocol included 5 min of cool down with 50 W load and 10 min of passive recovery in a seated position. Heart rate was recorded with an ANT+ heart rate monitor (Garmin, Olathe, USA), whereas respiratory parameters (i.e., oxygen uptake, exhaled carbon dioxide, and pul- monary ventilation) were measured with a breath-by-breath metabolimeter (Quark CPET, COSMED; Rome, Italy and Vyntus Vyntus CPX, Vyaire GmbH, Hochberg, Germany). According to the operating manufacturer’s instructions, the turbine flowmeter was calibrated using a 3-liter syringe. In addition, the gas analysis system was calibrated using room air: 21% O2, 0,03% CO2 and a certified gas mixture: 16% O2, 5% CO2 (Scott Medical ProductsTM, Plumsteadville, PA, USA) prior to each exercise test along with the delay and scrubber calibration. The maximal oxygen uptake, power, and heart rate value reached during the exercise test was considered as peak oxygen uptake (V̇O2peak), peak power, and peak heart rate (HRpeak). Biochemical measurements Participants were asked to arrive to the laboratory after 2 h fast. Three samples (3 ml each one) of peripheral venous blood were drawn using a vacutainer system. The first blood sample was drawn at baseline, the second was col- lected 15 minutes after reaching maximal exhaustion during the incremental test, and the final blood sample was taken approximately 24 hours later (Figure 1). Vacutainers were centrifuged at 10,000 rpm for 10 min and serum was separated and stored at –80°C for subsequent analysis. Samples were not diluted and were evaluated in duplicate in 40-well plates. Serum irisin was detected by ELISA Irisin kit (Cat. EK-067-29, Phoenix Pharmaceuti- cals, Burlingame, CA, USA) according to the manufacturer instructions, using the Victor Nivo multimode plate reader (PerkinElmer, Waltham, Massachusetts, USA). Inter- and intra-assay variation for irisin were <15% and <10%, re- spectively. All samples fell within the provided standard curve. Statistical analysis Using the G*Power software, the number of participants required was calculated to be 29, based on an effect size (ES) of 0.31,25 an α value of 0.05, and a desired statistical power (1-β) of 0.95. However, to account for potential drop- outs on the second day of evaluation, we conservatively re- cruited 34 participants. SPSS software (Version 27) was applied to perform statistical analysis. Normality of the data distribution was tested (Shapiro-Wilk test), data from total sample met the normality assumptions and the power anal- yses ensure a statistical power necessary to run parametric test. Therefore, one-way repeated measure analysis of vari- ance (ANOVA) was used to identify irisin changes from baseline, Post-hoc Contrasts were conducted to determine significant differences between means of time-points in the total sample. Regarding the data from the age groups, while normality was confirmed, the small sample size and asso- ciated lack of statistical power necessitated the use of non- parametric tests. Friedman-test for repeated measures was used to determine differences within group, Bonferroni cor- rection for pairwise comparison was used for data analysis. Wilcoxon signed rank test was performed to test differences within group in irisin variation (Delta). Mann-Whitney U test was used to test differences between groups (YA and MA). Pearson’s correlation was used to determine relation- ship between variables. Since age and some PE parameters differed between groups, partial correlation adjusted for age and peak power was performed to eliminate the strongest confounders. Variation irisin, absolute (Delta) and percent- - 179 - Figure 1. Schematic flowchart of the experimental protocol. Vacutainers indicate timing of blood sample collection, taken prior (baseline) to incremental exercise (V̇O2max test), immediately (15 min) and 24 h following the exhaustion. Rest indicates refraining from moderate and vigorous intensity, as well as long-duration training, in the 48 h prior to exercise and throughout the duration of the post-exercise study. (Created using BioRender.com). Non -co mmerc ial us e o nly Irisin release after an acute exercise Eur J Transl Myol 34 (2) 12693, 2024 doi: 10.4081/ejtm.2024.12693 age (Delta%), between baseline and post-exercise was com- puted (Delta=post-exercise irisin level – baseline irisin level), whereas the Delta% in irisin level after acute exer- cise was calculated using the following equation [(post-ex- ercise irisin level) − (baseline irisin level)/ baseline irisin level] x 100. Results Anthropometric characteristics, body composition, physical activity, and PE parameters of 34 participants (22 YA, 12 MA) are reported in Table 1. There were no significant dif- ferences between groups in anthropometric characteristics apart from percentage of FM that resulted higher for MA compared to YA. However, according to the ACSM nor- mative fitness categories for percentage of FM,23 the body composition was considered excellent in both groups. YA and MA were highly active26 showing a similar PAtot (P=0.943). Regarding PE parameters, significantly lower HRpeak and higher peak power were reached by MA during the exercise protocol, while no significant differences in rel- ative V̇O2peak were observed. When compared to the ACSM normative fitness categories for V̇O2max, 23 the cardiorespi- ratory fitness of YA was classified as good, while that of MA was classified as superior. Biochemical analysis revealed that irisin concentration 15 min and 24 h after the exercise was significantly higher than that detected at baseline in all subjects (P <0.001) (Figure 2a). Participants myokine levels increased (on average) by 15% (9.0±2.0 to 10.2±2.0 ng/ml) 15 min post-exercise, and by 55% (9.0±2.0 to 13.5±2.5 ng/ml) 24 h post-exercise compared with the baseline values. Both YA and MA showed significant increase in irisin concentration from baseline (YA, 9.7±1.7 ng/ml; MA, 7.6±1.6 ng/ml) to 15 min and 24 h post-exercise (YA, 11.1±1.8 ng/ml, P=0.003 and 14.5±2.2 ng/ml, P <0.001; MA, 8.7±1.5 ng/ml, P=0.025 and 11.8±2.2 ng/ml, P <0.001). Serum irisin was signifi- cantly higher in YA compared to MA for all measured time points (baseline P=0.002, 15 min P ≤0.001; 24 h P=0.005) (Figure 2b). However, MA individuals displayed a compa- rable change in serum irisin levels at both 15 minutes and 24 hours after exercise in comparison to their younger counterparts (Figure 2c). There was no significant correlation between baseline irisin levels and BMI (r=-0.034; P=0.848) or V̇O2peak (r=0.137; P=0.440). Circulating basal irisin was negatively correlated with FM (r= -0.471, P <0.005). However, this parameter did not withstand adjustment to age and peak power in par- tial correlation analysis (Table 2). Discussion This study reported increased irisin levels after an incre- mental exercise in 34 healthy male adults, providing origi- nal evidence that myokine levels enhance immediately after the end of exercise, but, interestingly, the highest concen- tration occurs one day after exercise. The incremental ex- ercise until exhaustion is a particularly effective stimulus for irisin production, allowing us to shed light both the - 180 - Figure 2. Circulation irisin concentration after an acute exercise. Serum irisin levels at baseline, 15 min and 24 h post-exercise in 34 participants (black bars) (a) and in two age-groups (b), young adults (YA) (white bars) and middle- aged adults (MA) (grey bars). Change in circulating irisin after the acute bout of incremental exercise in two age- groups, YA (white bars) and MA (grey bars) (c). P-values were obtained using ANOVA for repeated measured to compare baseline and post-exercise irisin levels (a). Mann- Whitney U test was used to verify differences between groups (b, c). Data are expressed as means±SD. **P <0.01; ***P <0.001. (Created by GraphPad). Non -co mmerc ial us e o nly Irisin release after an acute exercise Eur J Transl Myol 34 (2) 12693, 2024 doi: 10.4081/ejtm.2024.12693 quantity and time release of irisin in a homogeneous sample (all males), under well-defined pre- and post-exercise con- ditions. In literature, a previous study has failed to detect any changes in irisin expression after an acute bout of ex- hausting exercise.14 Only some authors documented modest changes of irisin levels immediately following (0-10 min) an acute bout of incremental exercise, 9,11-13 failing to detect post-exercise changes (after 60 and 180 min).12,13 Moreover, a meta-analysis has stated that irisin increased within 15 min, following an acute exercise.7 However, authors’ asser- tion was based on studies in which blood draws were taken immediately post-exercise except for one that collected post-exercise samples after 20 min. This time-limited data may have led Fox et al.7 to conclude that irisin increase is rapid and transient. Our findings clearly show that an acute bout of exhausting cycling induced a significant fast in- crease in irisin, but also demonstrate a long-drawn process. The fast increase in irisin concentration soon after exercise may be attributed to the proteolytic cleavage of available FNDC5 protein that is localized on the plasma membrane of muscle cells, leading to irisin release into the blood- stream in response to exercise. Differently, de novo FNDC5 production, induced by activation of PGC1-α expression, may require a long time (many hours), which could poten- tially explain the late elevation in circulating irisin reported in our study. The data reported by Norheim et al.27 appear to support this assumption. They investigated the effect of acute aerobic exercise on PGC1-α and FNDC5 (the irisin precursor) expression in skeletal muscle and they also eval- uated the circulating irisin levels in healthy adults, by col- - 181 - Table 1. Participants anthropometric characteristics, body composition, physical activity, and physical exercise parameters. Young adults Middle-aged adults P-value Age, yrs 24.6±3.5 54.6±5.7 <0.001 n 22 12 - Height, m 1.77±0.06 1.81±0.14 0.077 Weight, kg 73.1±9.8 76.53±7.3 0.387 BMI, kg/m2 23.2±2.4 23.4±2.2 0.773 FM, % 11.4±5.5 16.8±6.1 0.009 PAtot, METs-min/week 3733.6±3296.9 3101.4±1574.5 0.943 HRpeak, bpm 189±9 168±13 <0.001 V̇O2peak, ml/kg/min 49.0±9.8 44.8±5.1 0.256 Powerpeak, W 276±57 326±24 0.003 Table 2. Pearson’s correlations between baseline irisin levels, body composition, and physical exercise parameters in 34 participants Correlations Age- and Powerpeak-adjusted correla- tions Baseline irisin level (ng/ml) Baseline irisin level (ng/ml) r P r P BMI, kg/m2 -0.034 0.848 0.033 0.858 FM, % -0.471 0.005 -0.264 0.144 V̇O2peak, ml/kg/min 0.137 0.440 0.066 0.721 Non -co mmerc ial us e o nly Irisin release after an acute exercise Eur J Transl Myol 34 (2) 12693, 2024 doi: 10.4081/ejtm.2024.12693 lecting samples before, 0 min, and 2 h post-exercise. They observed that PGC1-α transcription was significantly in- duced after acute exercise, with a greater increment after 2 h post-exercise. As regard as circulating irisin levels, a tran- sient increase peaking immediately after acute exercise was found by the same authors, with a return to pre-exercise levels after 2 h rest. Consequently, the blood draws per- formed relatively early during the post-exercise period could have prevented the detection of further release of iri- sin which would occur in the following hours and that is likely due to new FNDC5 protein expression induced by PGC1-α. In a previous pilot study, we have observed that serum irisin level returns to baseline only after 48 h from the ending of an acute exercise bout to exhaustion.28 For this reason, in the present study, to all participants it has been required to abstain from moderate and vigorous intensity, and long-du- ration physical activity over the 48 h preceding the exercise. Whereas most of the studies reported only 24 h of rest9,13 or did not specify any information about it.11,12 Physical ac- tivities, carried out in the previous days, may lead to over- estimate basal myokines levels and contribute to a reduced increment in response to exercise. This provides important implications even for the design of chronic studies for which it appears necessary to measure basal irisin levels more than 24 hours after the last training session. Further- more, it is important to note that, except for Zugel et al.12 and Rodziewicz et al.,10 all the aforementioned studies in- cluded both sexes in the enrolled participants and have re- ported irisin levels of males and females in combined form. However, it is known that irisin levels are affected by sex11,17 and there are divergent responses to acute exercise in irisin concentration between males and females.29,30 Con- sequently, to exclude between group gender differences, males and females should be considered separately in irisin evaluation. This study enables us to observe the biological variations in this myokine accurately and without con- founding factors, albeit to a partial extent due to the absence of a female group. The second aim of this study was to compare irisin changes induced by acute bout of exercise in two different age groups. We demonstrated that the increase in circulating iri- sin was similar in both groups, stating, for the first time, the highest and massive change 24 h after a maximal effort. In- deed, the magnitude of average change in irisin was almost 55% that might be considered relevant from a physiological and clinical point of view. Moreover, we observed a com- parable increase about 15% (on average) of serum irisin even after 15 min post-exercise in both groups. These re- sults are in accordance with those reported by Huh et al.18 that have shown a similar increase (about 10%) in circulat- ing irisin immediately after 45-min of vigorous-to-exhaus- tive exercise in two age groups (young and older adults). While speculative, we can assert that despite aging, muscle cells of physically active older individuals maintain the same responsiveness after acute exercise compared to young individuals, thereby resulting in a similar exercise- induced increase in circulating irisin. In clear contrast to our observations, other researchers19-21 have observed no change in myokine levels after an exercise session in two age groups. It is likely that the intensity, as well as the type of exercise might have played a role in the blunted response of irisin levels to exercise stimulus. Future studies should further clarify the effect of nature, intensity, and duration of the exercise on irisin production. Moreover, we cannot exclude that the training level of the participants might have had a role in the irisin response to exhaustive exercise. To the best of our knowledge no studies have investigated the influence of training status in irisin response to acute exer- cise. Future studies should mandatorily explore the extent to which different levels of training can modulate the irisin response to exercise. Although our age groups showed a similar response of irisin to acute exercise, for each evaluated time point, we found that serum irisin was significantly higher in YA than MA. Our results are in accordance with some previous investi- gations,18,21 reporting higher basal irisin levels in the young compared to their older counterparts and in contrast with others.19,20 Finally, in the present study, we reported that circulating irisin is negatively associated with the percentage of FM. However, adjustment for age and peak power rendered this significant correlation null, suggesting that the latter may simply reflect the effect of age and cardiorespiratory fitness level. The findings of our research are subject to some limitations. First, it should be underlined that the present study involved a group of middle-aged healthy men with higher age-related cardiorespiratory fitness in comparison to the younger group, as evidenced by the higher levels of peak power achieved by MA during the maximal exercise test. For this reason, adjustment for peak power was used to eliminate this potential confounder. Second, the portion of subjects from each subgroup of the total sample was not the same and quite small. Finally, additional time point draws would allow to better outline the irisin time-course in response to an incremental bout of exercise and to identify more pre- cisely the irisin peak. Conclusions We have provided original evidence that an incremental test to exhaustion causes a relevant increase in circulating irisin one day after the ending of exercise across a wide age range and with a similar extent in both active young and middle- aged adults, likely providing pleiotropic and beneficial ef- fects on health. In our research, we used a maximal oxygen consumption test on a cycle ergometer as exogenous stimu- lus to increase irisin, however, maximal exercise to exhaus- tion is not a common daily training. Therefore, future research is needed to understand whether an increase in iri- sin levels can be achieved through lighter intensity activities or other types of exercise, that can be enjoyed by everyone. List of abbreviations PGC1-α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha FNDC5: Fibronectin type III domain-containing protein 5 - 182 - Non -co mmerc ial us e o nly Irisin release after an acute exercise Eur J Transl Myol 34 (2) 12693, 2024 doi: 10.4081/ejtm.2024.12693 BDNF: Brain-Derived Neurotrophic Factor UCP1: mitochondrial Uncoupling protein 1 YA: young adult group MA: middle-aged adult group BMI: body mass index FM: fat mass PAtot: total physical activity MET: metabolic equivalent of task V̇O2peak: peak oxygen uptake HRpeak: peak heart rate Powerpeak: peak power ANOVA: analysis of variance Contributions DT conceived and designed research; ET, SM, PV, CG, ABOS, ANM contributed to the acquisition of data; ET and ABON did the statistical analysis; ET wrote the first draft of the manuscript. All authors contributed to the analysis and interpretation of data, to revising the content of manu- script and they approved the final edited manuscript. Funding This work was supported by funds from PON ricerca e in- novazione 2014-2020 (D.M. 1061/2021). The publication of this article was funded by Università Cattolica del Sacro Cuore (Milan, Italy). Ethics approval The Ethics Committee of the Catholic University of the Sacred Heart approved this study (28-22). The study is con- formed with the Helsinki Declaration of 1964, as revised in 2013, concerning human and animal rights. Informed consent All patients participating in this study signed a written in- formed consent form for participating in this study. Patient consent for publication Written informed consent was obtained from a legally au- thorized representative(s) for anonymized patient infor- mation to be published in this article. Availability of data and materials All data generated or analyzed during this study are in- cluded in this published article. Conflict of interest The authors declare they have no financial, personal, or other conflicts of interest. Acknowledgments The authors are grateful to all volunteers involved in the study, and to MD Mario Toller, MD Alessandro Molinello, Dr Danny D’Agostini, Simone Mandelli, and Federico Parozzi for generous scientific/technical assistance. Corresponding Author Daniela Tavian Laboratory of Cellular Biochemistry and Molecular Bio- logy, CRIBENS, Università Cattolica del Sacro Cuore, Milan, Italy. Tel. 02/72348731. ORCID ID: 0000-0003-3333-0068 E-mail: daniela.tavian@unicatt.it Ester Tommasini ORCID ID: 0000-0003-2796-0688 E-mail: ester.tommasini@unicatt.it Sara Missaglia ORCID ID: 0000-0001-6551-6698 E-mail: sara.missaglia@unicatt.it Paola Vago ORCID ID: 0000-0002-0451-8144 E-mail: paola.vago@unicatt.it Christel Galvani ORCID ID: 0000-0002-0126-6033 E-mail: christel.galvani@unicatt.it Claudio Pecci ORCID ID: 0000-0002-5636-1943 E-mail: claudio.pecci@mapeisport.it Ermanno Rampinini ORCID ID: 0000-0002-9729-0862 E-mail: physiolab@mapeisport.it Andrea Bosio ORCID ID: 0000-0002-9772-4288 E-mail: andrea.bosio@mapeisport.it Andrea Morelli ORCID ID: 0000-0003-1753-7771 E-mail: andrea.morelli@mapeisport.it Andrea Bonanomi ORCID ID: 0000-0003-2857-1430 E-mail: andrea.bonanomi@unicatt.it Andrea Silvestrini ORCID ID: 0000-0002-2005-3746 E-mail: andrea.silvestrini@unicatt.it Alvaro Mordente ORCID ID: 0000-0003-3260-9796 E-mail: alvaro.mordente@unicatt.it - 183 - Non -co mmerc ial us e o nly mailto:daniela.tavian@unicatt.it Irisin release after an acute exercise Eur J Transl Myol 34 (2) 12693, 2024 doi: 10.4081/ejtm.2024.12693 References 1. 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Front Physiol 2018;9:1782. Disclaimer All claims expressed in this article are solely those of the authors and do not necessarily represent those of their af- filiated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. Submitted: 30 May 2024. Accepted: 20 June 2024. Early access: 2 July 2024. - 185 - Non -co mmerc ial us e o nly