Time Course of Muscle Damage and Inflammatory Responses to Resistance Training with Eccentric Overload in Trained Individuals

The purpose of this study was to observe the time course of muscle damage and inflammatory responses following an eccentric overload resistance-training (EO) program. 3 females (23.8 ± 2.6 years; 70.9 ± 12.7 kg; 1.6 ± 0.08 m) and 5 males (23.8 ± 2.6 years; 75.1 ± 11.2 kg; 1.8 ± 0.1 m) underwent thirteen training sessions (4 × 8–10 eccentric-only repetitions—80% of eccentric 1RM, one-minute rest, 2x week−1, during 7 weeks, for three exercises). Blood samples were collected prior to (Pre) and after two (P2), seven (P7), nine (P9), eleven (P11), and thirteen (P13) sessions, always 96 hours after last session. The reference change values (RCV) analysis was employed for comparing the responses, and the percentual differences between the serial results were calculated for each subject and compared with RCV95%. Four subjects presented significant changes for creatine kinase at P2, and another two at P13; six for C-reactive protein at P2, and three at P11; two for neutrophils at P2, P4, and P13, respectively; and only one for white blood cells at P2, P4, P7, and P9, for lymphocyte at P7, P9, and P13, and for platelet at P4. We conclude that EO induced high magnitude of muscle damage and inflammatory responses in the initial phase of the program with subsequent attenuation.


Introduction
Resistance-training protocols with eccentric overload (EO) have been investigated about their efficiency on strength and muscle cross-sectional area increases [1][2][3][4]. Experimental observations reported suggest that this training variation might be superior for both strength and hypertrophy development when compared to conventional concentric/eccentric training protocols [1][2][3]. EO is imposed once the maximal voluntary force is greater in this kind of muscle action [5,6], thus promoting a relatively lower workload for the eccentric phase during the conventional programs.
Meanwhile, the high-intensity unaccustomed eccentric component is characterized by inducing greater muscle damage incidence with signi�cant acute decreases in muscle function [7], acute-phase in�ammatory responses [8][9][10][11][12], and increases in plasmatic activity of myo�brillar proteins such as creatine kinase, lactate dehydrogenase, and myoglobin [12][13][14][15]. Instead, there have also been reports that a bout of eccentric exercise at several weeks interval results in a marked reduction in the symptoms associated with muscle damage [9]. is adaptation of eccentric exerciseinducing protection against subsequent tissue damages has been referred as repeated bout effect [16].
Observing the data on the initial drop in performance induced by the eccentric activity [7,15,17], recent researches have aimed at a very important aspect of the physical training process: the monitoring of biochemical and immunological markers and also the performance [18][19][20]. High training loads with insufficient recovery periods have been suggested Mediators of �n�ammation to induce overreaching and overtraining in team sport players [18,21], and the monitoring of these responses along with the training period may be critical for the identi�cation of such events.
Hematological and biochemical analyses are oen used to identify fatigue and recovery in athletes during the training seasons [18], although relatively few studies have systematically examined the use of EO resistance-training protocols for monitoring such factors [2][3][4]12]. To compare individual blood parameter values with reference intervals obtained from a physically active population may also be a useful tool to monitor the adaptive effects of exercise. However, this form of comparative analysis has certain limitations, as laboratory results may be in�uenced by biological variation [22], hindering the clinical results interpretation, particularly those from consecutive analysis performed for the same individual.
Considering this aspect to compare the serial results, in the present study, we adopted the reference change values (RCV) or critical difference analysis. e RCV is a tool employed to verify if the difference between two consecutive samples is signi�cant and biologically relevant, considering the various components of intrinsic variation affecting laboratory assays. ese components include factors related to laboratory activity (preanalytical and analytical variation) and those related to normal intraindividual biological variation [23]. As such, the RCV de�nes the percentage of change that should be exceeded when considering the analytical and biological variation inherent to a test, in order to evaluate signi�cant changes between two consecutive measurements.
Bearing this in mind, the aim of this study was to observe the time course of muscle damage and in�ammatory responses to resistance-training with EO adopting the RCV to compare the serial results. Our initial hypothesis was to observe high magnitude of muscle damages on the initial phase of the program, together with greater in�ammatory responses. However, a subsequent attenuation of these events by the repeated bout-effect occurrence was expected [9,16].

Experimental
Design. irteen training sessions were performed twice a week, performed at the same hour of the day, and under the supervision of the researchers involved. One familiarization session with the resistance exercise equipments happened three weeks before the program started. Blood samples were collected in eight distinct time points throughout the program and always before the individuals started the sessions. ese time points were Pre (prior to the �rst session), P2 (aer two sessions), P� (aer seven sessions), P9 (aer nine sessions), P11 (aer eleven sessions), and 96 hours aer the last training session (P13).

Participants.
Eight healthy subjects (3 female: age: 23.8 ± 2.6 years; body mass: 70.9 ± 12.7 kg; height: 1.6 ± 0.08 m; % body fat: 29.6 ± 4.3; and 5 male: age: 23.8 ± 2.6 years; body mass: 75.1 ± 11.2 kg; height: 1.8 ± 0.1 m; % body fat: 20.0 ± 4.9) participated in the study. e inclusion criteria included being engaged in resistance-training programs for at least one year, and no intake of exogenous anabolicandrogenic steroids, drugs, medication, or dietary supplements with potential effects on physical performance is recorded. Training programs usually performed by the subjects consisted of 3-5 sets of 6-12 repetitions with 1-2minute rest interval between sets, performed 4-5 times per week. Subjects gave written informed consent aer being advised about the purposes and the risks associated with the study. Subjects fasted for one hour prior to the blood collection, being also required to refrain from strenuous exercise and the consumption of alcohol, tobacco or caffeine 48 hours before the testing sessions. During the period of the study, none of the participants reported any kind of infection state that could affect his/her immune responses despite the in�ammation induced by the exercise. Research Ethics Committee (019/2004) approved the experimental protocol.

Strength Tests. e one repetition maximal test (1RM)
was employed a fortnight prior to the beginning of the study, aiming the prescription of the resistance-training intensity (% 1RM). Since the present study makes a distinction between the concentric and eccentric phase of the movement, it was necessary to employ a speci�c test to access the maximum weight to be bore by the eccentric-only muscle action. e typical 1RM test re�ects only the maximum weight that can be lied using a concentric action-designated as 1RMcon. is speci�c evaluation is referred as 1RM eccentric (1RMecc) [24], and it is adopted due to the observations that skeletal muscles are capable of developing much higher forces when they contract eccentrically compared to concentrically [24]. e 1RMcon testing was conducted using the methods described by Brow and Weir [25], and for the 1RMecc the methods described by Hollander and coworkers [24]. ere were three-to-�ve single trials for the validation of the test, which was the subject using proper form and completing the entire li in a controlled manner without assistance [26].

Training
Program. e program consisted of thirteen sessions over seven weeks, performed on Tuesdays and ursdays, always at the same time of the day and under the supervision of the researchers involved. Prior to each session, subjects completed a standardized warm up program of static stretching exercises, and a speci�c warm up of 8 repetitions with approximately 50% of the estimated 1RMcon for each exercise. Training protocol consisted of 4 sets of 8-10 eccentric-only repetitions with 80% of 1RMecc, and one minute of rest between sets. e concentric phase of the movement was performed with the assistance of the researchers, and the exercises employed were Bench Press, 45-degree Leg Press, and Bent-Over Rows.

Blood
Samples. Blood samples were collected under standardized conditions: 2.0 mL of total venous blood was collected in vacuum tubes containing EDTA/K3 to determine hematological parameters, and 8.0 mL of venous blood was collected in tubes with a Vacuette (Greiner Bio-one) gel separator in order to obtain serum for biochemical measurements. Blood samples were collected 96 hours aer the last Mediators of In�ammation 3 training bout, in the morning aer 12 hours of fasting, in a seated position, transported at 4 ∘ C to the laboratory within 30 minutes, centrifuged under refrigeration at 1.800 ×g for 10 minutes, immediately separated, and protected from light.
Biochemical measurements were conducted with commercial kits (Wiener Lab; Rosario, Argentina) and with an Autolab (Boehringer, Mannheim, Germany) analyzer. e assay included creatine kinase (CK) activity, and C-reactive protein (CRP). To minimize analytical variations, the same technician tested all samples without changing reagent lots, standards, or control materials.

Discussion
Our main �ndings were that four subjects presented sig-ni�cant changes for CK at P2, and other two at P13. For CRP six subjects presented signi�cant changes at P2, one at P4, other at P9, three at P11, and other at P13. Only two subjects presented signi�cant changes for NEUTR. Subject 7 presented changes at P2 and P4, and subject 6 at P13 only. In addition, subject 7 also presented signi�cant changes for WBC at P2, P4, P7 and P9, for LYNF at P7, P9, and P13, and for PLT at P4. No signi�cant changes were observed for RBC, PLT, Ht, MCH, MCHC, RDW, MCV, and Hb. No signi�cant correlations were observed between CK and CRP as well as WBC and NEUTR at P2.
To the best of our knowledge, this is the �rst study to employ the RCV speci�cally for physically active subjects when comparing the temporal behavior of in�ammatory and muscle damage responses to EO. Individual blood analyses provided by the RCV allowed the identi�cation of the speci�c subjects seeming to be more susceptible for in�ammatory processes and muscle damage. Conventional statistical analysis considers the probability of occurrence of one event for a group and not for speci�c subjects, what may mask or disregard the more responsive individuals. ese results appear to be very important for future studies aiming at monitoring these events.
CK responses presented by four subjects at P2 supported our initial hypothesis to observe high magnitudes of muscle damage on the initial phase of the program. e subsequent attenuation of this event con�rmed the occurrence of the repeated bout effect, with the exception of two subjects with signi�cant increases of CK at P13 in relation to RCV, but within the RI for physically active subjects (Table 1). e repeated bout effect refers to the adaptation whereby a single bout of eccentric exercise protects against muscle damage T 1: Signi�cant changes in CK and CRP and the speci�c subjects presenting alterations. RI: reference interval for physically active subjects [23]; Δ: difference between two consecutive analyses.
T 2: Signi�cant changes in NEUTR, WBC, LYNF, and PLT and the speci�c subjects presenting alterations. RI: reference interval for physically active subjects [23]; Δ: difference between two consecutive analysis. from subsequent bouts [16]. e potential adaptations that explain the phenomena have been categorized as neural, mechanical, and cellular. Regarding the cellular adaptations there is evidence of longitudinal addition of sarcomeres and adaptations in the in�ammatory response following an initial bout of eccentric exercise, limiting also the proliferation of damage. �espite four individuals not presenting signi�cant increases in CK at P2, we cannot completely state that they did not experience muscle injuries. Evidences in the literature report that CK plasma activity is not considered as a goldstandard evaluation of muscle damage because it does not present a signi�cant linear correlation with muscle functions and ultrastructural changes in muscle following exercise [17,30,31]. Presently, muscle functions measurements are considered the most indicated methods for quantifying injuries because the event results in an immediate and prolonged reduction in these parameters, persisting over the entire span of the progression of the degenerative and regenerative processes [17,32]. On the other hand, plasma activities of myo�bril proteins are not evidenced over this entire time course of degenerative and regenerative processes, and even when evidenced, they may or may not be correlated with the magnitude of the functional decrements [17,32]. We Mediators of In�ammation 5 recognize that one limitation of the present study was not having a quanti�cation of muscle functions parallel to plasma CK activity in order to establish possible correlations between them. CRP responses do not correlate to CK, emphasizing that elevated CRP levels may be associated to damage in nonskeletal muscle tissue.
Similar to CK, CRP responses were characterized by two time points when subjects appeared to express higher responses. Despite the similarity of response at P2, no signi�cant correlations with CK were found, con�rming the observations made by previous studies [13]. Literature emphasizes that elevated CRP levels may simply mark the clearance of modi�ed molecules or necrotic tissue in an effort to limit damage associated with the in�ammatory process [33]. Providing a valid prediction of the progression of vascular lesions in the absence of acute infection, the increases in CRP levels may be associated to damage in nonskeletal muscle tissue [33], thus, explaining the poor correlation between CK and CRP responses found. It is important to observe that all subjects presented signi�cant changes in relation to RCV but within the traditional RI for physically active subjects (Table 1).
Time course of muscle damage and in�ammatory responses have also been investigated in two recent studies [34,35]. e �rst study evaluated the process following an international rugby union game. An acute-phase in�ammatory response re�ected through immediate increases in serum cortisol and IL-6, followed by delayed increases in serum CK (14 hours) activity and CRP (38 hours) was observed. e �ndings suggested that a rugby match elicits disturbances in host immunity, which last up to 38 hours into the recovery period. e second research observed the process together with performance changes, following a soccer match (in the morning of the game day, immediately aer, and 24, 48, 72, 96, 120, and 144 hours aer match). Performance deteriorated 1-to-4 days aer match, an acutephase in�ammatory response consisting of a postmatch peak of leukocyte count, 24-hour peak of CRP, and 48-hour peak of CK were observed.
According to the behaviors reported in the aforementioned studies, it becomes evident that the moment for collection of blood samples is a crucial aspect for their observation (i.e., the in�ammatory process appears to have an acute-phase response with a subsequent attenuation). e moment of blood collection in the present study was 96 hours aer the last training bout, which could not be the most adequate one for the observation of the time course for some markers. ese observations may help us to explain the reasons why only two subjects presented signi�cant changes for NEUTR at P13, and only one for WBC (at P2, P4, P7, and P9), LYNF (at P7, P9, and P13), and PLT (at P4). Other subjects may have expressed an early-stage (24-48 hours aer last bout) acute-phase in�ammatory responses, which was not detected 96 hours aer the last bout.
Speci�c resistance-training in�ammatory responses were reported by Simonson and Jackson [11]. Blood samples were drawn at before, aer, 15 minutes aer, and 30 minutes aer exercise. All leukocyte subpopulations, except for basophils and eosinophils, increased at aer exercise but the counts declined at aer, 15, and 30 minutes aer exercise. Only NEUTR did not return to preexercise levels by 30 minutes aer exercise. e majority of resistance exercise-induced leukocytosis was due to an increase in circulating LYNF and monocytes. In the study, the authors suggested that by the lack of large alterations and rapid recovery from cell number, resistance training is not immunosuppressive. Meanwhile, we highlight that the data of the aforementioned studies were not analyzed according to the RCV for physically active subjects.

Conclusions
EO resistance training protocol induced high magnitudes of muscle damage and CRP responses on the initial phase of the program, with a subsequent attenuation of the event. Such behavior con�rms the occurrence of the repeated bout effect for this particular training protocol, except for two subjects. Nevertheless, CRP responses do not correlate to CK, emphasizing that elevated CRP levels may be associated to damage in nonskeletal muscle tissue.

Con�ict of �nterests
e authors declare that they have no con�ict of interests.