Regarding general neuromuscular health, resistance exercise (RE) is considered the most efficient method for enhancing muscular strength, power, and muscular endurance (American College of Sports Medicine, 2009), triggering multiple benefits with numerous positive outcomes evident in the literature (Garber et al., 2011). However, the beneficial effects of resistance exercise can be influenced by exercise selection, exercise order, intensity, number of sets, and resting interval between sets (American College of Sports Medicine, 2009). Modulation of such parameters determines specific adaptations toward increases in strength or hypertrophy (Borde et al., 2015; Schoenfeld et al., 2019).

In particular, regarding modulation of inter-set lengths, evidence suggests that either shorter or longer inter-set duration (within a range of 60 s to 3 min) may promote muscle hypertrophy (Grgic et al., 2017), while longer rests (usually 3 min or longer) seem to be more beneficial for strength development (de Salles et al., 2009). Therefore, it is still not clear which mechanisms mediated by inter-set length may determine hypertrophic adaptations. Previous studies indicated that differences in resting intervals between sets may independently influence the repetition performance (Senna et al., 2011, 2016; Scudese et al., 2015; Matos et al., 2021), as well as promote changes in neuromuscular (Willardson and Burkett, 2008), endocrine (Scudese et al., 2016), cardiorespiratory (Ratamess et al., 2007), and even immune responses to the exercise session (Gerosa-Neto et al., 2016; Rossi et al., 2016).

During RE sessions, the muscular tissue is exposed to micro-injury (which occurs after concentric and eccentric contractions, with greater damage observed after eccentric exercise) (Markus et al., 2021). Exercise-induced muscle damage (EIMD) has been mainly observed in the sarcolemma, cytoskeleton, and contractile elements of the muscle. Despite EIMD may have short-term (24–48 h) detrimental effects on muscle function, due to pain and inflammation, it is thought that the associated muscle inflammation promotes increased protein turnover, which may lead to long-term adaptations (Schoenfeld, 2012; Fukada et al., 2020). In particular, short-inter-set lengths (60 s or below) are seen to promote greater muscle damage than longer rest periods (3 min or above) (Machado and Willardson, 2010) and acutely increase anabolic hormonal levels (Henselmans and Schoenfeld, 2014). EIMD also increases the expression of both pro and anti-inflammatory cytokines of local muscle tissue (Calle and Fernandez, 2010; Minari and Thomatieli-Santos, 2021). Modulation of genetic muscle regulators and activation of muscle satellite cells, mediated by different anti-inflammatory cytokines, may be one of the major factors that lead to the regeneration or even hypertrophy of muscle tissue (Peake et al., 2006). However, muscle damage and inflammatory response derived from RE are still not completely elucidated and might play a key role regarding the physiological mechanism related to RE adaptations (Spiering et al., 2008; Margaritelis et al., 2021).

Research has indicated that once a tissue is damaged, similar increases in biochemical markers occur as a consequence of the extravasation of the content of the damaged cells, despite different stimuli are provided (Rodrigues et al., 2010; Evangelista et al., 2011; Machado et al., 2012). Increases which also occur after acute bouts of high-intensity interval training [increases observed in Interleukin 6 (IL-6), Interleukin 10 (IL-10), and tumor necrosis factor α (TNF-α); Gerosa-Neto et al., 2020]. After muscle damage occurs, an increase in leukocyte recruitment to the bloodstream is observed as an attempt to reverse the general breakdown (Kilgore et al., 2002; Fatouros and Jamurtas, 2016) due to the process of repair and tissue remodeling (Raastad et al., 1985).

Changes in the hemodynamic profile driven by mechanical stimuli, promote increased sympathetic action with release of catecholamines during exercise. These appear to be an important factor driving the elevation of leukocyte concentration, promoting the cellular recruitment of lymphoid organs and endothelium to the blood by mechanical loading (Pedersen and Hoffman-Goetz, 2000).

Consequently, different cytokines have effects on the mobilization of neutrophils from the bone marrow to the blood, increasing the leukocyte count beyond the patterns presented immediately at the end of an exercise session (Pedersen and Hoffman-Goetz, 2000), thereby triggering a second phase increase in cell types. This physiological cascade of events that characterizes the immune system profile occurs at different time-point peaks that may not immediately fall after the exercise (Mayhew et al., 2005; Chatzinikolaou et al., 2010). Although the general inflammatory response is vastly clinically studied, little is known between the immune system response related to modulation of RE parameters, as different rest intervals between sets (Mayhew et al., 2005). Few experiments have attempted to elucidate the behavior of this process by analyzing the biomarkers of tissue damage, leukocyte profile, and cytokine expression of pro and anti-inflammatory action after RE performed with different intervals between sets (Rodrigues et al., 2010; Evangelista et al., 2011; Machado et al., 2012; Rossi et al., 2016). Therefore, the purpose of this study was to analyze the effects of two distinct rest period lengths (1 and 3 min, which are typically used in hypertrophy or strength protocols, respectively) between RE sets of equalized volume on muscle tissue damage [CK and Lactate Dehydrogenase (LDH)] and inflammatory biomarkers responses.

Our key findings regard the muscle tissue damage (CK) triggered by the shorter inter-set length (1-min) observed from the early 12 h to the 24 h post-exercise window, compared to the 3-min rest condition. Increased muscle damage contributed to the increases observed in the total number of leukocytes, neutrophils (3–12 h post-exercise), and monocytes (12 h post-exercise) in the shorter inter-set rest condition. Altogether, increased inflammatory process due to increased muscle damage, increased pro-inflammatory cytokines (IL-1β, TNF-α, and GM-CSF) mainly after 6 and 12 h. Following the aforementioned increase, an increase in anti-inflammatory cytokines (IL-5, IL-6, and IL-10) was observed (6, 12, and 24 h). In accordance with these findings, the shorter rest period length triggered significantly higher RPE values compared to the longer rest condition for both volume-equated training protocols.

The level of serum CK and LDH can be elevated due to muscle tissue damage as a consequence of high-intensity training (Rodrigues et al., 2010). These increases may be a response to metabolic and mechanical stimuli (Brancaccio et al., 2007). One of the mechanisms may be related to local tissue damage with degeneration and sarcomeric fragmentation between the Z discs (Brancaccio et al., 2007). Another mechanism that seems to be associated with this damage is metabolic exhaustion of muscle fibers, which in turn presents a decrease in membrane resistance after an elevation in free internal calcium ions, as well as the activation of the potassium channels, occurred by the reductions in cellular energy reserves (Fink and Luttgau, 1976). Therefore, the response pattern of these enzymes (CK and LDH) suggests an appropriate indicator of the intensity of a previously performed exercise (Bessa et al., 2016). Therefore, it is plausible to interpret that different manipulations in RE variables can trigger distinct biological responses (Helms et al., 2020). Nevertheless, other studies have indeed focused on the impact of rest interval manipulation on muscle tissue damage and immune responses (Rodrigues et al., 2010; Evangelista et al., 2011; Machado et al., 2012; Gerosa-Neto et al., 2016; Rossi et al., 2016). However, these present great variations in blood time-point choices, populations, non-equalized vs. equalized volume and exercise schemes, relative loads, and nutritional controls. All this variability, unfortunately, prevents a consensus regarding post-exercise muscle tissue damage and immune responses patterns in acute response to distinct inter-set lengths.

Rodrigues et al. (2010) compared CK and LDH concentrations at multiple post-exercise time-points (>24 h) after RE sessions with different inter-set intervals. The two experimental sessions consisted of three sets with loads of 80% of 1-RM until concentric failure, which caused a 24% larger volume for the longer interval (non-equalized method). There were no significant differences in CK and LDH concentrations at any post-exercise assessment between the different intervals. Other authors observed the effects of different rest intervals on CK and LDH in sessions with 10-RM loads until concentric failure (Machado et al., 2011). The intervals stipulated in this experiment were 1-, 1.5-, 2-, and 3-min between sets and exercises. The CK and LDH were significantly elevated after 24 and 72 h in all sessions and without differences between the distinct intervals. When comparing these studies (Rodrigues et al., 2010; Machado et al., 2011) with the present experiment, there is an evident methodological difference, mainly at the proposed attempt to analyze the interval time between sets without any control of other variables as volume or intensity.

It is important to underline, that in practical terms different rest periods are chosen to allow full (longer interest periods) or incomplete recovery (shorter interest periods) between sets. In the case of longer rest periods, this avoids muscle impairments, with the consequence to counteract load decreases caused by fatigue (Schoenfeld et al., 2016), which may occur if using interest length shorter than 2 min (Matos et al., 2021). Despite perceived exertion may result increased with lower-rest intervals (Scudese et al., 2015), a progressive load decrease along with subsequent sets determines a decrease in both total volume and intensity (Matos et al., 2021), which could result in lower gains in terms of hypertrophy and strength (Schoenfeld et al., 2015, 2019).

In another investigation (Evangelista et al., 2011) which compared the differences between two distinct rest interval lengths (1- vs. 3-min) on volume, muscle damage, and muscle soreness, the subjects performed an experimental protocol consisting of three sets of biceps exercise with 40% of maximal voluntary contraction. The results showed that individuals who performed the exercises with the longer rest intervals performed more volume (as expected). Still, there were no differences for CK (24 and 48 h after exercise) and muscle soreness between groups, probably due to the low intensity performed. Similarly, Machado et al. (2012) examined the values of CK activity after RE sessions in subjects that the authors classified as possessing high, medium, or low responsiveness to the exercises. Individuals classified with high and medium responsiveness demonstrated an elevated CK activity after the 1-min interval session compared to the 3-min interval session. However, it is important to note that the blood examinations were restricted to 24 and 48 h post-exercise, and the training routine consisted of only one single-joint exercise (bicep curl).

By analyzing the previous studies’ methodology, it is safe to state that our experiment is original regarding the training volume equalization and the meticulously tailored timing of CK and LDH blood examinations. More specifically, our results allowed to observe that the shorter inter-set period potentiated the muscle damage markers responses. It should also be noted that the main focus of the present study was the initial and rapidly (up to 24 h) curve identification of CK and LDH responses, demonstrating an early post-exercise sensibility of those biomarkers. Moreover, the CK increase had its onset as early as 6 h post-exercise for the 1-min interval, and the LDH showed its peak around the 12 h post-exercise assessment. These data bring to light new evidence that the majority of the previous investigations might have missed the sensitivity window for those responses.

Regarding the pro- and anti-inflammatory process resulting from the RE responses, shortly after the muscle damage occurrence, there was leukocyte mobilization followed by its migration to the injured muscle tissue (Peake et al., 2006). The neutrophils and monocytes, the cellular types mobilized in greater magnitude at different moments in the process of muscle tissue remodeling, are responsible for the reduction of the damaged muscular tissue and the release of pro- and anti-inflammatory cytokines.

Several studies on the inflammatory responses to RE (Peake et al., 2006; Phillips et al., 2010) showed that the total training volume (rather than intensity) directly influences pro-inflammatory interleukins up to 12 h after training. Smith et al. (2000) and Hirose et al. (2004) observed important modifications for pro-inflammatory interleukins in assessments performed up to 72 h after exercise even with low training volumes. Despite evidence exists regarding manipulation of RE volume, little is known regarding inter-set differences and inflammatory responses. An investigation by Kraemer et al. (1996) in 1996 examined the impact of exercise-induced circulating plasma cortisol elevations and leukocyte counts with different inter-set lengths. Venous blood samples were obtained pre, during exercise, and 5-min after exercise. There were no significant variations in leukocyte differential counts. Probably, due to the very close assessment point, 5 min post-exercise. Though, seeking to determine the effect of distinct rest intervals on leukocyte levels during moderate-intensity RE, Mayhew et al. (2005) performed a study where nine men completed exercise sessions with 1 vs. 3 min inter-set intervals. Blood was collected at rest, immediately after, 60-, and 90-min post-exercise, and the leukocyte concentration was analyzed. Increased lymphocytosis and monocytosis were observed after the 1-min inter-set length protocol but not after the 3-min inter-set length protocol. Although the studies by Kraemer et al. (1996) and the more recent of Mayhew et al. (2005) have verified different inter-set length and their responses to leukocyte count, important limitations, such as the timing of the blood sample collection may have compromised the outcomes retrieved. In the present study, the leukocyte count was aligned with the CK and LDH assessments during the process of tissue damage (up to 24 h after exercise). In such consideration, possibly after muscle damage in response to high-intensity RE, occurred an important leukocyte mobilization and invasion (mainly neutrophils) to the injured tissue alongside with a significant monocyte elevation.

More recently, a two-part experiment (Gerosa-Neto et al., 2016; Rossi et al., 2016) aimed to verify the influence of a very short (30-s) and moderate (90-s) inter-set length on performance, inflammatory, and metabolic responses in healthy adults and recreational weightlifters. In part 1, eight healthy subjects performed two exercises at 70% of 1-RM; and in part 2, the procedures were repeated; however, with 90% of 1-RM. Both conditions each with the different inter-set lengths. The TNF-α, IL-6, and IL-10 were assessed at baseline, immediately after exercise, 15 and 30 min post-exercise. The authors concluded that exhaustive and heavy strength exercises conducted with different inter-set length decreased performance; however, an augmented inflammatory and metabolic response were found only in the longer 90-s interval. Despite similarities in inter-set length are observed when comparing these studies to our experiment, differences occur in the timing of the post-exercise blood assessments and the volume equalization.

Other finding of this investigation, is that related to perceived exertion of the participants. We demonstrated significant increases in RPE with the progression of the sets for both exercises, with the highest values found for the shorter 1-min inter-set length. To date, only two investigations compared different rest conditions on the RPE responses in submaximal exercises, both observing that the shorter rests, increased RPE responses (Scudese et al., 2015, 2016). Other experiments focused on the RPE influences of different rest intervals between sets in distinct types of exercise (Senna et al., 2011) and near-maximum loads zones (Scudese et al., 2015; Senna et al., 2016).

Limitations to our study are the relatively small sample size, since not many participants who met all the required inclusion criteria were willing to participate due to the repeated blood sample collection. Further, our evaluation up to 24 h post-exercise only is a further limitation as muscle damage and inflammation typically occur up until 48 h post-exercise. However, it was aim of this study to evaluate the curve response within the first 24 h. The inclusion of a third 5-min inter-rest condition could have provided further confirmation regarding the data included for the 3-min inter-rest length. Further consideration could be that the intensity used across the two protocols was 85% of 10RM, which may not be of sufficient intensity to produced muscle damage with long rest periods and may have produced different responses compared to repetition maximum training.

This study brings a new approach to the way muscle damage and inflammatory response are investigated after a resistance training routine. Our results suggest that when equalized for training volume, a shorter inter-set length promotes a considerably greater damage to muscle tissues, as well as a longer duration of the inflammatory process of this tissue. Based on these findings, specifically on the durable inflammatory augmentation observed with the shorter inter-set length, practitioners with the intent to promote hypertrophy or local muscle endurance development should consider implementing shorter inter-set lengths when using a similar type of training method. Other important aspect to consider is that shorter inter-set periods allow shorter workouts, which in turn improve training efficiency.

This data might contribute to future recommendations focused on different goals requiring considerable muscle tissue damage and inflammatory response for its optimization. These results are applicable and limited to the specific exercises, inter-set length, and load examined. However, we strongly recommend that future studies should evaluate distinct exercise schemes, other load ranges, types of rest period (active vs. passive), and whole-body training sessions.

The raw data supporting the conclusions of this article will be made available by the authors, after reasonable request.

The studies involving human participants were reviewed and approved by the Federal University of State of Rio de Janeiro ethics committee (CAAE 63803717.2.0000.5285). The patients/participants provided their written informed consent to participate in this study.

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES)–Finance Code 001.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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