European Journal of Applied Physiology

, Volume 113, Issue 10, pp 2577–2586

Effect of cryotherapy on muscle recovery and inflammation following a bout of damaging exercise

Authors

  • Naomi J. Crystal
    • Robert Kertzer Exercise Physiology LaboratoryUniversity of New Hampshire
    • Department of KinesiologyUniversity of New Hampshire
  • David H. Townson
    • Department of Molecular, Cellular, and Biomedical SciencesUniversity of New Hampshire
  • Summer B. Cook
    • Robert Kertzer Exercise Physiology LaboratoryUniversity of New Hampshire
    • Department of KinesiologyUniversity of New Hampshire
    • Robert Kertzer Exercise Physiology LaboratoryUniversity of New Hampshire
    • Department of KinesiologyUniversity of New Hampshire
Original Article

DOI: 10.1007/s00421-013-2693-9

Cite this article as:
Crystal, N.J., Townson, D.H., Cook, S.B. et al. Eur J Appl Physiol (2013) 113: 2577. doi:10.1007/s00421-013-2693-9

Abstract

The purpose of this study was to determine the effect of cryotherapy on the inflammatory response to muscle-damaging exercise using a randomized trial. Twenty recreationally active males completed a 40-min run at a −10 % grade to induce muscle damage. Ten of the subjects were immersed in a 5 °C ice bath for 20 min and the other ten served as controls. Knee extensor peak torque, soreness rating, and thigh circumference were obtained pre- and post-run, and 1, 6, 24, 48, and 72 h post-run. Blood samples were obtained pre- and post-run, and 1, 6 and 24 h post-run for assay of plasma chemokine ligand 2 (CCL2). Peak torque decreased from 270 ± 57 Nm at baseline to 253 ± 65 Nm post-run and increased to 295 ± 68 Nm by 72 h post-run with no differences between groups (p = 0.491). Soreness rating increased from 3.6 ± 6.0 mm out of 100 mm at baseline to 47.4 ± 28.2 mm post-run and remained elevated at all time points with no differences between groups (p = 0.696). CCL2 concentrations increased from 116 ± 31 pg mL−1 at baseline to 293 ± 109 pg mL−1 at 6 h post-run (control) and from 100 ± 27 pg mL−1 at baseline to 208 ± 71 pg mL−1 at 6 h post-run (cryotherapy). The difference between groups was not significant (p = 0.116), but there was a trend for lower CCL2 in the cryotherapy group at 6 h (p = 0.102), though this measure was highly variable. In conclusion, 20 min of cryotherapy was ineffective in attenuating the strength decrement and soreness seen after muscle-damaging exercise, but may have mitigated the rise in plasma CCL2 concentration. These results do not support the use of cryotherapy during recovery.

Keywords

Eccentric exerciseDownhill runInflammationChemokine ligand-2

Abbreviations

CCL2

Plasma chemokine ligand 2

DOMS

Delayed-onset muscle soreness

MVC

Maximal voluntary contraction

VASS

Visual analog scale for soreness

Introduction

Cryotherapy, commonly performed by way of ice baths, is a popular post-exercise recovery modality utilized by athletes and clinicians to reduce inflammation and speed recovery (Barnett 2006; Cheung et al. 2003; Connolly et al. 2003). In the context of this paper, cryotherapy is defined as immersion in water below 15 °C (Bleakley and Davison 2010). While there have been investigations of the effects of cryotherapy on inflammation and recovery from damaging exercise (Eston and Peters 1999; Howatson et al. 2009; Ingram et al. 2009; Jakeman et al. 2009; Lane and Wenger 2004; Paddon-Jones and Quigley 1997; Rowsell et al. 2009; Sellwood et al. 2007; Vaile et al. 2008), there is no clear consensus on the effectiveness of this treatment modality, despite its widespread use.

Exercise induces an inflammatory response proportional to the duration, intensity, and mass of muscle used as well as the damage resulting from the exercise (Camus et al. 1993; Giraldo et al. 2009). To study muscle impairment and inflammation in a laboratory setting, eccentric exercise is commonly used as a means of inducing muscle injury. Running, particularly downhill running, has a large eccentric component and produces significant myocyte damage and inflammatory response (Buford et al. 2009; Malm et al. 2004; Smith et al. 2007).

Myocyte damage is manifested by delayed-onset muscle soreness (DOMS), strength and power decrements, as well as an increase in plasma concentrations of proteins normally found within the muscle cell (e.g., creatine kinase) (Best and Hunter 2000). An acute inflammatory response ensues following this damage including the release of inflammatory cytokines (Best and Hunter 2000). The inflammatory response is accompanied by symptoms such as swelling and soreness, which individuals may seek to attenuate with cryotherapy (Best and Hunter 2000). The inflammatory response to downhill running (Hubal et al. 2008; Peake et al. 2005) and other eccentric exercises is well characterized (Malm et al. 2000), and has recently been reviewed by Tidball (2005).

An inflammatory cytokine of interest is chemokine ligand 2 (CCL2) because it is a sensitive marker of inflammation that increases after running (Peake et al. 2005). Furthermore, it shows reduced levels after a second session of exercise, indicating that it may be involved in the repeated bout effect, or adaptation to eccentric exercise (Smith et al. 2007). CCL2 serves to recruit monocytes to the damaged tissue so they can phagocytize cellular debris and facilitate the rebuilding process (Chazaud et al. 2009). Research in CCL2-deficient mice has demonstrated that the cytokine is necessary for the recruitment of monocytes for phagocytosis and stimulates production of insulin-like growth factor 1 (IGF-1) for repair (Lu et al. 2011; Shireman et al. 2006). Although it would negatively impact adaptation, a reduction in CCL2 may be beneficial for short-term recovery in that it would attenuate secondary damage caused by the inflammatory process and the associated DOMS, thus potentially mitigating the performance decrement.

Though immune cells repair exercise-induced muscle damage, they also exacerbate the injury by releasing reactive oxygen species (ROS) that oxidize molecules within the myocyte (Best et al. 1999; Brickson et al. 2001). This results in an increase in the noticeable symptoms of muscle damage including DOMS and strength decrement. Because these symptoms interfere with subsequent competition and training performance, elite and recreational athletes often seek strategies to minimize muscle damage and speed recovery (Cheung et al. 2003). Recovery strategies such as stretching, massage, light activity, and cryotherapy are purported to reduce inflammation, diminish soreness, and facilitate a more rapid return of performance capabilities (Ingram et al. 2009; Lane and Wenger 2004; Lapointe et al. 2002; Tidball and Wehling-Henricks 2007; Vaile et al. 2008). Speeding the recovery process is especially beneficial when applied to occasions when an athlete must perform with little recovery time between competitions. It is also valuable for the general exerciser, as unaccustomed exercise causes soreness and loss of function which can interfere with daily activities and subsequent exercise bouts.

Studies investigating cryotherapy as a recovery modality have used a range of immersion protocols and have measured different markers of muscle damage making it difficult to assess efficacy. Some studies have found cryotherapy to be effective in reducing the symptoms of muscle damage such as swelling, soreness, sprinting performance impairment, cycling time trial and interval performance impairment, and elevated plasma creatine kinase and C-reactive protein (Eston and Peters 1999; Ingram et al. 2009; Lane and Wenger 2004; Rowsell et al. 2009; Vaile et al. 2008). However, other studies indicate that cryotherapy has no effect on performance, soreness, swelling, plasma creatine kinase, or lactate dehydrogenase measures (Howatson et al. 2009; Jakeman et al. 2009; Paddon-Jones and Quigley 1997; Rowsell et al. 2009; Sellwood et al. 2007). Small sample sizes, small effect sizes, and high variability of measures among subjects may have prevented statistical significance in these studies, yet it is plausible that cryotherapy is ineffective at aiding muscle recovery. Subsequently, there is no agreement on the effectiveness of cryotherapy as an exercise recovery modality.

The purpose of this study was to examine the impact of cryotherapy on the inflammatory response to downhill running, and muscle recovery over 3 days, by analyzing plasma levels of CCL2 as well as the more commonly studied soreness, swelling, and isometric strength variables. It was hypothesized that cryotherapy would reduce the inflammatory response to downhill running, specifically that cryotherapy would attenuate the rise in CCL2 normally observed following downhill running, ameliorate DOMS, reduce swelling in the thigh, and minimize knee extensor strength decrement.

Methods

Subjects

Twenty males (mean ± SD: age 21.2 ± 2.3 years; height 1.78 ± 0.05 m; mass 76.4 ± 9.6 kg; VO2peak = 58.9 ± 8.6 ml kg−1 min−1), who were unaccustomed to cryotherapy, were recruited to the study via flyers around the university and by word of mouth. Subjects were then randomly assigned to the cryotherapy or control group (n = 10 per group). Subjects were classified as recreationally active from a self-reported physical activity questionnaire. We defined “recreationally active” as meeting the American College of Sports Medicine’s guidelines for cardiovascular exercise, that is, 30 min of moderate intensity (3–5.9 METs) exercise 5 days per week or 20 min of vigorous (≥6 METs) exercise 3 days per week, but not exceeding 7 h of vigorous exercise per week. Subjects reported a diverse history of training which included recreational exercise, as well as previous participation in high school and college athletics, but at the time of the study all met our definition of recreationally active. Subjects also reported a variety of activities in which they routinely engaged including running, team sports, strength training, cycling, and cross-country skiing. Subjects were excluded from the study for having any musculoskeletal injuries that interfered with running, having Reynaud’s disease or cold allergy, or regularly using anti-inflammatory medication. One subject withdrew from the study 5 min into the downhill run and an additional subject was added in his place. The University of New Hampshire’s Institutional Review Board approved the use of human subjects in accordance with the Belmont Report, and written informed consent of each subject was obtained prior to their participation.

Experimental design

The study was a randomized clinical trial that evaluated the effect of cryotherapy (independent variable) on the inflammatory cytokine CCL2, isometric knee extensor strength, perceived soreness, and swelling (dependent variables). These dependent variables were chosen as they are markers of muscle damage, they are commonly reported symptoms of eccentric exercise, they may impact physical performance, and they are reasons for which individuals would use cryotherapy. The study took place from October to June. Subjects visited the laboratory six times and a timeline summary of the study can be seen in Fig. 1. Visit 1 consisted of anthropometric measurements, a peak oxygen consumption (VO2peak) test, determination of downhill running speed, and familiarization sessions with the strength assessment and visual analog scale for soreness (VASS). Visit 2 took place an average of 9 ± 6 days after the first visit and each session began between 11:00 a.m. and 1:00 p.m. to account for diurnal variation. A blood draw for assay of CCL2, and baseline strength, thigh circumference, and soreness data were collected prior to the downhill run. Subjects then completed a 40-min downhill run to induce muscle damage and all measures were recorded again post-run. The cryotherapy (ice bath) or control condition was completed immediately after post-run measures were taken and all measures were recorded again at 1, 6 h (Visit 3), and 24 h (Visit 4) after completion of the downhill run. At 48 h (Visit 5) and 72 h (Visit 6) after the run, only strength, thigh circumference, and soreness measures were collected as CCL2 was expected to return to baseline by 24 h post-exercise (Smith et al. 2007).
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Fig. 1

Timeline of the study. Pre-run immediately before the downhill run, Post-run immediately after the cooldown following the downhill run

VO2peak testing and determination of individual downhill running speed

During Visit 1 a modified Balke protocol was used to determine VO2peak during treadmill running (Quinton, Q65, Seattle, WA, USA). Subjects began running at a moderate running pace (between 2.7 and 3.8 m s−1 based on self-reported fitness level) at 0 % grade; then the grade was increased 1 % per minute until volitional exhaustion. Continuous respiratory measurements were recorded using a SensorMedics Vmax Metabolic Measurements Cart (CareFusion Corporation, San Diego, CA, USA). Data were recorded breath by breath and averaged over 30 s for analysis. Heart rate was monitored with a Polar™ heart rate monitor (Polar Electro, Lake Success, NY, USA) and recorded at the end of each minute. Ratings of perceived exertion (RPE) on the 6–20 point Borg scale were recorded at the end of each stage.

To determine the appropriate intensity for the downhill runs, 60 % of VO2peak was calculated for each participant. The familiarization session with downhill running also served as a test to determine the treadmill speed necessary to achieve an intensity of 60 % VO2peak while running at a −10 % grade on the treadmill, and was completed during Visit 1. Subjects began jogging at 1.7–2.5 m s−1 on a level treadmill and the grade was gradually lowered to reach a −10 % grade. Oxygen consumption data were collected and speed was increased until oxygen consumption reached 60 % of VO2peak. This speed was recorded and used during the downhill run on Visit 2. Subjects ran downhill for no longer than 5 min during the habituation session to avoid inducing DOMS and a repeated bout effect.

Exercise protocol

Subjects were instructed to refrain from any vigorous exercise for at least 72 h before the downhill run and until after the 72 h post-run visit. Subjects were also asked to refrain from the use of anti-inflammatory drugs and all supplements for 2 weeks prior to the downhill run and until after the 72 h post-run visit. Compliance was assessed by a written questionnaire that was completed at the beginning of each visit. During Visit 2, muscle damage was induced in all subjects by a 40-min downhill treadmill run (Gaitway Treadmill, Kistler, Amherst, NY, USA), at −10 % grade, at the speed corresponding to 60 % of the subjects’ VO2peak. The 40-min downhill run was a novel activity for all subjects and was therefore expected to induce significant DOMS. At completion of the run, subjects were given a 3-min level cooldown walk at a self-selected pace on the treadmill.

Cryotherapy

Ten of the subjects were randomly assigned to the cryotherapy treatment and ten to the control condition and were informed of their group assignment prior to the downhill run. Before subjects entered the tank, water temperature was adjusted by adding ice until it reached 3–5 °C. Immediately following this post-run assessments on Visit 2, each of the cryotherapy subjects stood quietly in the tank of 5 ± 2 °C water that came to the top of the thigh for a duration of 20 min. Water temperature was checked an average of two times during the treatment and more ice was added if needed. The temperature did not rise above 7 °C during the 20-min treatment for any subject. Each of the control subjects stood quietly in the laboratory for the same period of time following his post-run assessments for consistency with previous studies that have used no treatment as the control condition (Eston and Peters 1999; Howatson et al. 2009; Ingram et al. 2009; Jakeman et al. 2009; Lane and Wenger 2004; Paddon-Jones and Quigley 1997).

Swelling and soreness

Circumference of the non-dominant thigh was measured with a tension-controlled tape measure (Creative Health Products, Ann Arbor, MI, USA) at the midpoint of a line drawn from the anterior superior iliac spine to the superior pole of the patella. Thigh circumference has an intraobserver percentage of reliability coefficient >0.98 % and technical error of measurement averages 0.67 cm (Moreno et al. 2003). Soreness experienced while walking down a flight of stairs was self-reported using a VASS. The VASS is an unmarked horizontal 100-mm line with the terminal descriptors “no soreness” and “very, very sore”. Subjects were instructed to “think about how your legs feel” while walking down the stairs and then marked their perceived soreness on the line. Their pain score was the distance in millimeters from the “no soreness” end of the line to the subject’s mark. The intraclass correlation coefficient for a visual analog scale has been reported to be rxx = 0.97 when used for experimental heat pain and chronic pain; however, this study used it for soreness (Price et al. 1983). Soreness and swelling measurements were taken prior to the run (baseline), immediately after the cooldown, and 1, 6, 24, 48, and 72 h post-exercise.

Strength

Maximal isometric knee extensor torque of the non-dominant leg was measured at 105o of knee extension (with 180o being full extension) on a HUMAC Norm dynamometer (CSMI, Stoughton, MA, USA) and recorded with a BIOPAC MP150 data acquisition system (BIOPAC Systems, Inc, Goleta, CA, USA). Participants were seated with a hip angle of 85o and pushed maximally against the resistance arm for 3 s. The mean of the peak torque obtained from three attempts was recorded and used for analysis. Isometric testing of the knee extensors using this dynamometer has been shown to have an intraclass correlation coefficient of rxx = 0.95 for peak isometric torque (LaRoche et al. 2008). Measurements were taken prior to and immediately after the downhill run and 1, 6, 24, 48, and 72 h post-exercise.

Chemokine ligand 2

Venous blood samples from an antecubital vein were drawn prior to and immediately after the downhill run and 1, 6, and 24 h post-run. Two milliliters of blood was drawn using a standard venipuncture into vacutainer tubes containing sodium heparin. Samples were centrifuged for 15 min at 3,000×g immediately after collection. Plasma was aliquotted and stored at −80 °C until assayed. CCL2 was quantified using human CCL2 Quantikine ELISA kits according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Briefly, plasma samples were incubated in a 96-well microplate pre-coated with monoclonal antibody to human CCL2. The plate was subsequently washed with washing buffer, leaving only the cytokine bound to antibodies. Each sample was then exposed to horseradish peroxidase-linked polyclonal antibody specific for CCL2. Following another washing step, the wells were exposed to substrate to produce a colorimetric precipitate proportional to the amount of CCL2 in the sample. The color reaction was terminated and the optical density of each sample was determined at 450 nm using a microplate reader (Biotek ELx808, BioTek Instruments, Inc., Winooski, VT, USA). Cytokine concentrations for the samples were determined by comparison to a standard curve of known concentrations of cytokines and their optical density. All samples were run in duplicate. The intra-assay coefficient of variation was 4.6 % and the inter-assay coefficient of variation was 3.7 %.

Statistical analyses

Estimates of skewness and kurtosis were used to screen for normality of the data. Homogeneity of variance was assessed using Levene’s statistic and Mauchley’s test of sphericity. For variables that violated the assumption of homogeneity of variances, a Greenhouse–Geisser correction factor was applied to the degrees of freedom and subsequent corrected p values were reported in the results. Baseline comparisons were made between groups for descriptive statistics, CCL2, strength, thigh circumference and soreness, using a one-way analysis of variance (ANOVA). To compare the differences in CCL2 between groups over time following the downhill run, a 2 × 5 (group × time) repeated-measures ANOVA was used. A separate 2 × 7 (group × time) repeated-measures ANOVA was used to compare differences in strength, thigh circumference, and soreness. The significance level for all testing was set at P < 0.05. Data are reported as means ± SD. Effect sizes for the cryotherapy treatment were determined from the ANOVA by partial eta squared (η2).

Results

All data were normally distributed except reported walking time and VASS pre-run. One subject reported that he walked 200 min each day which is unusually high. VASS at pre-run was skewed to the right because pre-run soreness ratings abutted zero. All variables met the homogeneity of variance assumption except MVC and CCL2. The Greenhouse–Geisser adjusted p values are reported for these two variables.

Subject demographics

There were no significant differences between the cryotherapy and control groups for demographics, reported activity level, VO2peak, downhill running speed, or any of the baseline measurements (Table 1). All subjects completed the 40-min run at 60 % of their VO2peak without stopping.
Table 1

Comparison of sample characteristics

 

Control group

Cryotherapy group

p value

Age (years)

21.5 ± 3.2

20.9 ± 0.9

0.575

Mass (kg)

75.2 ± 9.7

77.6 ± 9.9

0.589

Height (m)

1.76 ± 0.05

1.80 ± 0.04

0.075

Body mass index (kg m−2)

24.3 ± 2.5

24.1 ± 3.2

0.835

Activity level

 Running distance (km week−1)

14.7 ± 12.1

18.5 ± 16.7

0.689

 Walking time (min week−1)

265 ± 124

366 ± 372

0.693

 Moderate intensity activity (min week−1)

249 ± 127

323 ± 271

0.441

 Vigorous intensity activity (min week−1)

175 ± 165

190 ± 154

0.830

VO2peak (mL kg−1 min−1)

58.1 ± 8.1

59.7 ± 9.3

0.561

Downhill running speed (m s−1)

3.68 ± 0.86

3.55 ± 0.55

0.425

Downhill run distance (km)

8.83 ± 2.07

8.52 ± 1.34

0.694

Values are the mean ± SD

Maximum voluntary contraction torque

The muscle-damaging downhill run resulted in a significant change in maximum voluntary contraction torque (MVC) over time (p < 0.001, power = 1.000). Torque decreased by an average of 6.2 % immediately following the downhill run and this decrement persisted at 1 h post-run. Over the next 2 days, torque recovered to pre-exercise values and by 72 h post-run torque had increased significantly above baseline values by 9.2 % (Fig. 2a). The response to the downhill run over time explained 35 % of the variation in MVC (η2 = 0.35). There was no significant difference in MVC between the cryotherapy and control groups (p = 0.992, power = 0.346, η2 = 0.0). The interaction between group and time was not significant and explained little of the variance in MVC (p = 0.491, power = 0.346, η2 = 0.03), leaving 62 % of the variance unexplained.
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Fig. 2

Peak voluntary torque and soreness over time. a Peak knee extensor isometric torque over time. b Perceived soreness over time. Values are the mean ± SD. *Time effect, significantly different from pre (p < 0.05). #Time effect, significantly different from post (p < 0.05)

Soreness

The downhill run elicited a significant change in soreness over time (p < 0.001; power = 1.00). The run caused an average increase in VASS rating from 3.7 to 47.4 mm out of 100 mm which remained significantly elevated above baseline at all subsequent time points. Soreness declined at the 6-h mark then increased again to peak at 56.4 mm 24 h post-run and finally declined to 22.2 mm by 72 h post-run (Fig. 2b). Sixty percent of the variation in the VASS ratings can be attributed to the time at which the measures were taken. The cryotherapy and control groups did not differ in their VASS ratings (p = 0.256; power = 0.199), and only 3.5 % of the variation in VASS ratings was due to group. Both groups showed similar responses over time (p = 0.696; power = 0.246) with only 1.3 % of the variation being explained by the group × time interaction.

Thigh circumference

The downhill run did not alter thigh circumference measurements over time (p = 0.151; power = 0.597; η2 = 0.033). The cryotherapy and control groups did not differ in thigh circumference (p = 0.677; power = 0.069; η2 = 0.584), nor were there differences in how the groups responded over time (p = 0.860; power = 0.170; η2 = 0.009). For example, thigh circumference was 54.1 ± 4.2 cm post-run for the control group and 55.0 ± 3.8 cm for the cryotherapy group with no change at 24 h post-run (54.2 ± 4.4 and 54.8 ± 3.9 cm, respectively), or at any other time point.

Chemokine ligand-2

Data for plasma CCL2 concentration were available for 16 subjects (n = 9 cryotherapy group; n = 7 control group); plasma from the first four subjects of the study thawed as a result of a freezer malfunction and were discarded. The muscle-damaging protocol had a significant effect on CCL2 concentrations over time (p < 0.001; power = 1.000). Average plasma CCL2 concentrations increased from 108 to 156 pg mL−1 post-run, peaked at 251 pg mL−1 6 h post-run, and declined to 119 pg mL−1 by 24 h post-run (Fig. 3a). Fifty-nine percent of the variation in CCL2 concentrations was attributed to time, while only 6.2 % was attributed to group and 4 % to group × time interactions, leaving 30 % of the variance in CCL2 unexplained (Fig. 3b). There was no significant effect of cryotherapy on CCL2 concentrations (p = 0.116; power = 0.344; η2 = 0.062). Groups did not differ in their plasma CCL2 concentrations as a function of time (p = 0.430; power = 0.217; η2 = 0.041). However, there was a trend toward lower CCL2 concentrations in the cryotherapy group at 6 h post-run. The peak in CCL2 concentration at 6 h post-run was 173 % higher than post-run in the control group and only 146 % higher than post-run in the cryotherapy group (control group increased from 170 to 293 pg mL−1, while the cryotherapy increased from 143 to 208 pg mL−1).
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Fig. 3

Plasma chemokine ligand 2 (CCL2) over time. a Comparison of CCL2 concentration between cryotherapy and control groups over time. b Comparison of the individual percent change of CCL2 from the post-run (pre-treatment) time point. Values are the mean ± SD. *Time effect, significantly different from pre (p < 0.05). #Time effect, significantly different from post (p < 0.05)

Discussion

This study explored the effects of cryotherapy on the CCL2 response to damaging exercise and contributes to previous work on strength, swelling, and soreness measures. Important findings include a significant effect of time for strength, soreness, and for plasma measures of CCL2 following the downhill run, of which the time response for CCL2 was previously unknown. This study demonstrated no significant therapeutic effect of cryotherapy on any of the variables measured, although there was a trend toward lower CCL2 concentration following cryotherapy.

Downhill running is an ideal model for studying cryotherapy because it induces DOMS and an inflammatory response. Running is a part of training for many sports, and runners in particular tend to utilize cryotherapy (e.g., ice baths) as a method to speed recovery. As expected, 40 min of running down a −10 % grade at 60 % of VO2peak induced muscle damage and inflammation. Muscle damage was suggested by the strength decrement that did not recover until about 48 h post-run, and the significant DOMS which remained elevated at 72 h post-run. The observed increases in plasma CCL2 concentrations post-run are typical of an inflammatory response associated with muscle-damaging exercise (Peake et al. 2005).

Strength, swelling, and soreness

Cryotherapy was not effective at attenuating the strength decrement observed after the downhill run. None of the variation in MVC was due to cryotherapy (0.0 %), and only 3.1 % was due to the group × time interaction, suggesting that this recovery modality had little effect on reducing strength decrement following muscle-damaging exercise. A lack of effect of cryotherapy on strength was also observed by other researchers (Howatson et al. 2009), though one found a non-significant 25 % attenuation of the strength decrement (Eston and Peters 1999). Others have observed improvements in other performance variables such as sprint performance (Ingram et al. 2009) and cycling power output (Vaile et al. 2008) following cryotherapy. Perhaps, performance of sport-specific activities is improved with cryotherapy, but isometric strength is not, because performing functional movements depends on the ability to move through a normal range of motion, which is compromised with DOMS (LaRoche and Connolly 2006). The increase in strength seen in both groups at the 72-h time point is likely a result of familiarization with the isometric strength test.

Any swelling that may have occurred in the thigh as a result of the run was not detectable with the circumference measurement used nor was any difference resulting from cryotherapy detectable. Most studies agree that cryotherapy does not significantly affect swelling, specifically circumference as measured with an anthropometric tape measure (Howatson et al. 2009; Sellwood et al. 2007) or volume measured via water displacement (Paddon-Jones and Quigley 1997).

VASS increased dramatically in response to the run. An interesting finding is the double peaks in soreness that occurred in both groups immediately post-run and again at 24 h post-run. We believe the first peak represents the acute fatigue-related pain that occurred immediately following exercise and the peak at 24 h post-run likely represents DOMS. At 6 h post-run, acute pain had declined significantly, and possibly at a greater rate in the cryotherapy group (note the slopes of the lines from 1 to 6 h in Fig. 2b), but DOMS had not yet set in causing a temporary drop in soreness ratings. There were, however, no significant differences between groups over time in muscle soreness. Others who used the VASS to measure the influence of cryotherapy on soreness have also failed to observe any effect. For example, Jakeman et al. (2009) did not detect a difference in soreness using the VASS at five time points over 96 h, following ten sets of ten counter movement jumps (Jakeman et al. 2009). Similar to our study, Sellwood et al. (2007) observed no effect of cryotherapy (three 1-min immersions in 5 °C water) on soreness when rated on a 100-mm VASS at 24, 48 or 72 h post-eccentric quadriceps exercise (Sellwood et al. 2007). Researchers who measured muscle soreness using applied pressure also reported no effect of cryotherapy (Eston and Peters 1999). The results of this study do not support the common use of cryotherapy in ameliorating soreness.

However, a few researchers have found a decrease in soreness when cryotherapy was used during recovery. Ingram et al. (2009) observed a significant reduction in soreness at 24 h in those who underwent two 5-min immersions in 10 °C water when soreness in the quadriceps was rated on a ten-point Likert scale (Ingram et al. 2009). Rowsell et al. (2009) also observed a reduction in soreness at 24, 48, and 72 h throughout a soccer tournament in those who underwent five, 1-min immersions in 10 °C water after each match when subjects rated leg soreness on a scale of 1–10 (Rowsell et al. 2009). One possible explanation for the different results is that the coefficient of variation for the ten-point Likert scales used in the previous studies is lower than for the 100-mm VASS used in this study (23.5 and 27.6 versus 65.5 %). Thus, differences in the soreness measure used, cryotherapy protocol, exercise performed, time of the measurements, and subject sample confound the effects of cryotherapy on muscle soreness, necessitating additional study.

Plasma CCL2

Plasma CCL2 concentrations followed the pattern expected after muscle-damaging exercise with a 2–2.5-fold increase by the 6-h post-run mark. Of the five time points at which blood was drawn, CCL2 concentration was highest at the 6-h point. It is possible that the true peak may have occurred before or after 6 h at a time when blood was not drawn. This study is one of the first to quantify the time course of CCL2 after muscle-damaging exercise and showed a trend toward a reduction in CCL2 concentration with cryotherapy. Peake et al. (2005) found a marked increase in CCL2 immediately, and 1 h after downhill running, but did not obtain plasma measures at any later time points, making our study the first to measure CCL2 over a 24-h time period.

While the majority of variation in CCL2 concentration was based on differences between subjects, cryotherapy accounted for 6.2 % of the variation in CCL2 and the group × time interaction accounted for 4.1 % of the variation. At the pre-exercise time point, the between-subject variation in CCL2 was not unusual for plasma markers of inflammation and muscle damage. The mean CCL2 concentration at this point of the study was 108 pg mL−1, with an SD of 29 pg mL−1, which elicited a coefficient of variation of 27.2 %. To put this in perspective, the coefficient of variation for creatine kinase (a common marker of muscle damage) at baseline in previous cryotherapy studies was 45.1, 69.4 and 60.0 % (Howatson et al. 2009; Ingram et al. 2009; Rowsell et al. 2009). Similar to the highly variable changes of creatine kinase observed in previous studies, the individual change of CCL2 after exercise was inconsistent between individuals in both groups in this study (Fig. 3b). Some subjects experienced more than a 200 % increase in CCL2 from the post-run to the 6-h time point, whereas others had minimal change. This individual variability in the inflammatory response contributes to the difficulty of assessing the efficacy of cryotherapy treatment. Unfortunately, it is not possible to definitively identify the source of the individual variation in CCL2, but the volume, intensity, and type of previous physical activity could modify the reaction, as could differences in the responsiveness of individuals’ immune systems.

Although the differences between the groups did not reach statistical significance, CCL2 was 29 % lower in the cryotherapy group at 6 h post-exercise. This finding should be interpreted with caution as the response was highly variable among subjects, especially within the control group where one individual experienced a particularly high CCL2 peak. As this is a new area of research, the magnitude of change in CCL2 that is clinically significant is not yet known, but CCL2 deficiency has been shown to impair skeletal muscle regeneration in mice (Shireman et al. 2006). CCL2 is needed to recruit monocytes into injured muscles to conduct phagocytosis and produce IGF-1 for injury repair. CCL2 signaling also up-regulates IGF-1 expression by intramuscular macrophages to promote skeletal muscle repair (Lu et al. 2011). Thus, reducing CCL2 following exercise may negatively impact adaptation. However, limiting the rise in CCL2 may be beneficial for short-term recovery, in that it may lessen the secondary damage caused by the inflammatory process and the associated DOMS. This could possibly minimize performance loss in the short term, though we did not observe an effect on maximal voluntary isometric strength.

The study did not support the hypothesis that cryotherapy reduces the inflammatory response to downhill running as measured by plasma CCL2, DOMS, swelling in the thigh, or decrement of knee extensor strength. There were no significant differences between the groups over time for any of the dependent variables. This raises the question of whether or not cryotherapy is effective at reducing the symptoms of muscle damage. The effect sizes (η2) for group main effects and group × time interactions in this study were very small for soreness, thigh circumference, and strength, suggesting cryotherapy had almost no effect on these measures. In fact, to detect differences, sample sizes of 75–1,000 subjects would have been necessary for these variables. Conversely, 21 subjects would have been necessary to detect a significant difference in plasma CCL2 concentration, suggesting cryotherapy may have an effect on this inflammatory cytokine. Although the current study contained a limited number of subjects, relative differences in CCL2 were observed due to time and treatment effects. However, additional study is needed to more fully evaluate the merit of cryotherapy in attenuating the inflammatory response and hastening recovery from muscle-damaging exercise.

Future work should clarify the effects of cryotherapy on CCL2 and other cytokines in clinical populations, as well as in recreational and elite athletes, with both accustomed and unaccustomed exercises. If cryotherapy is shown to be effective at speeding recovery, studies should determine if there is an optimal protocol. Equally important are the long-term effects of cryotherapy on adaptation when it is used regularly. Because the inflammatory response is important in mediating adaptation to exercise (Best and Hunter 2000; Brunelli and Rovere-Querini 2008; Butterfield et al. 2006; Chazaud et al. 2009), attenuating this response may reduce training adaptations in those who chronically uses cryotherapy. For example, Yamane et al. (2006) found that cryotherapy reduced improvements in unilateral VO2max and vascular adaptations following training. This suggests that cryotherapy may not benefit long-term fitness when used regularly. Perhaps, a more effective application of cryotherapy would be multi-day events when athletes must perform repeatedly with little rest, and the benefits of quick recovery outweigh the risk of attenuated long-term adaptation.

Limitations

This study used recreationally active men performing an unaccustomed downhill running exercise, which may limit the generalizability of the results. A number of factors were not studied that may influence the inflammatory response and recovery such as diet, rest, lifestyle, and additional fitness parameters. Isometric MVC torque was used to measure strength, but a sport-specific measure such as sprinting time may have demonstrated greater decrements in performance. Variability in plasma CCL2 concentration over time necessitates a larger sample size for the attainment of statistical significance between groups.

Conclusion

Forty minutes of downhill running at 60 % of VO2peak induces muscle damage and inflammation, but the results of the current study do not support the use of cryotherapy as a recovery modality. Plasma CCL2 concentration, as a marker of inflammation, increased immediately following downhill running, peaked at 6 h post-exercise and returned near baseline by 24 h post-exercise. Implementation of 20 min of cryotherapy at 5 °C was not effective at attenuating the loss of strength and increase in soreness seen after muscle-damaging exercise, but may have mitigated the rise in plasma CCL2 concentration.

Acknowledgments

The authors would like to thank the subjects who participated in this study. This study was conducted with no external funding.

Conflict of interest

The authors report no conflict of interest.

Copyright information

© Springer-Verlag Berlin Heidelberg 2013