Accepted Manuscript: 10.1016/j.physbeh.2018.06.008

Accepted Manuscript
Lymphocytes and monocytes egress peripheral blood within

minutes after cessation of steady state exercise: A detailed
temporal analysis of leukocyte extravasation
Bridgette V. Rooney, Austin B. Bigley, Emily C. LaVoy, Mitzi

Laughlin, Charles Pedlar, Richard J. Simpson
PII: S0031-9384(18)30317-2
DOI: doi:10.1016/j.physbeh.2018.06.008
Reference: PHB 12227
To appear in: Physiology & Behavior
Received date: 21 March 2018
Revised date: 29 May 2018
Accepted date: 6 June 2018
Please cite this article as: Bridgette V. Rooney, Austin B. Bigley, Emily C. LaVoy,
Mitzi Laughlin, Charles Pedlar, Richard J. Simpson , Lymphocytes and monocytes egress
peripheral blood within minutes after cessation of steady state exercise: A detailed
temporal analysis of leukocyte extravasation. Phb (2017), doi:10.1016/
j.physbeh.2018.06.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.
ACCEPTED MANUSCRIPT
Lymphocytes and monocytes egress peripheral blood within

minutes after cessation of steady state exercise: a detailed
temporal analysis of leukocyte extravasation
T
Bridgette V. Rooney1, Austin B. Bigley1,3, Emily C. LaVoy1, Mitzi Laughlin1, Charles Pedlar2,
Richard J. Simpson1,3,4,5,6
IP
CR
1
University of Houston, Department of Health and Human performance, Houston, TX
US
2
School of Sport, Health and Applied Science, St. Mary’s University, Twickenham, UK
3
University of Arizona, Department of Nutritional Sciences, Tucson, Arizona
AN
4
University of Arizona, Department of Pediatrics, Tucson, AZ
5
University of Arizona, Department of Immunobiology, Tucson, AZ
M
6
University of Texas MD Anderson Cancer Center, Department of Behavioral Sciences,
Houston, TX
ED
Correspondence to:
PT
Richard J. Simpson, PhD FACSM
Associate Professor
CE
Department of Nutritional Sciences
Department of Pediatrics
AC
Department of Immunobiology
The University of Arizona
1177 E. Fourth Street
Room 308, Shantz Building
Tucson, Arizona 85721, USA
Tel: 520-621-4108
Email: rjsimpson@email.arizona.edu
ACCEPTED MANUSCRIPT
Abstract:
Acute exercise evokes an almost instantaneous lymphocytosis, followed by sustained
lymphopenia that occurs within just 30-60 minutes after exercise cessation. The aim of this
study was to characterize the immediate (order of minutes) post-exercise kinetics of lymphocyte
and monocyte egress, and to determine whether this egress is associated with heart rate
recovery following a single bout of steady state dynamic exercise. Eleven healthy subjects
cycled for 30-minutes at ~70% of their estimated peak power. Blood samples were collected
from an intravenous catheter before exercise, during exercise (E) at +15 and +30 minutes, and
during passive recovery (R) at exactly +1, +2, +3, +4, +5 and +10 minutes after exercise
T
cessation. Complete blood counts and flow cytometry were used to enumerate total monocytes,
lymphocytes: CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, NK-cells and  T-cells in whole blood.
IP
Both lymphocytes and monocytes displayed rapid egress kinetics, by R+3 the total numbers of
all cell types examined were significantly lower than E+30. NK-cells egressed more rapidly than
CR
other lymphocyte subtypes, followed by CD8+, , and then CD4+ T-cells. Further, the egress of
NK-cells, CD4+, and CD8+ T-cells positively correlated with heart rate recovery after exercise
cessation. In conclusion, lymphocyte and monocyte egress is rapid and occurs within minutes of
US
exercise recovery, underscoring both the importance of collection time for post exercise blood
samples, and the use of intravenous catheters to capture peak cell mobilization. The rate of
egress may be dependent on how quickly hemodynamic equilibrium is restored on cessation of
AN
exercise and is, therefore, likely to be influenced by individual fitness levels.
M
ED
PT
CE
AC
Key Words: Exercise Immunology, Leukocytosis, T-Cell, NK-Cell, Neutrophilia,

Trafficking, Lymphopenia
ACCEPTED MANUSCRIPT
1.1 Introduction:
A single bout of dynamic exercise causes an almost instantaneous mobilization of leukocytes
into the peripheral circulation [1,2]. Neutrophils account for the vast majority of this exercise-
induced leukocytosis, although lymphocytes [3] and monocytes [4] also mobilize in relatively
large numbers. During exercise recovery, the circulating neutrophil count may continue to rise
[3], while lymphocyte numbers rapidly decline. Indeed, the circulating lymphocyte count may fall
by 30-50% below resting levels within the first hour of exercise recovery, sometimes resulting in
a transient clinical lymphopenia (<1.0 x106/ml) that may persist for up to 6h [1,2,5]. The extent
by which lymphocytes are redeployed between the blood and tissues with exercise is largely
T
influenced by the intensity and duration of the bout. In the major lymphocyte subtypes, NK-cells
exhibit the largest relative redeployment in response to exercise, followed by  T-cells, CD8+
IP
T-cells, CD4+ T-cells and, lastly, B-cells [6,7]. Furthermore, the NK-cell and T-cell subtypes that
are preferentially redeployed with exercise tend to have: greater cytotoxic and effector functions
CR
[8–11], heightened expression of adrenoceptors [7] and glucocorticoid receptors [12,13] and
phenotypes associated with tissue migration [14], and antigen experience [15,16].
US
The mechanisms responsible for leukocyte redeployment in response to exercise are
multifaceted, but are largely due to changes in hemodynamic shear stress and the actions of
catecholamines and glucocorticoids [17]. Increases in cardiac output and heart rate amplify
hemodynamic forces along the vascular endothelium and at target organ reservoirs, resulting in
AN
the demargination of adherent leukocytes from vessel walls [18] and from the pulmonary [19],
hepatic, and splenic pools [20]. Additionally, either through the reduction in adhesion molecule
expression or the obstruction of adhesion molecule receptor/ligand binding, catecholamines
M
promote leukocytes to circulate freely [21]. Glucocorticoid secretion, resulting from the activation
of the hypothalamic-pituitary-adrenal (HPA) axis, also influences leukocyte trafficking [13,22].
ED
Where the mobilized cells traffic to following their egress from the peripheral blood compartment
is not well understood. They may return to their pre-exercise location, or might migrate to
areas/tissues that now require their sentinel and/or reparative action following activation of the
biological stress response [17].
PT
Although it is known that blood lymphocyte numbers can fall below pre-exercise levels within the
first 30-60 minutes after exercise cessation [5], the kinetics of lymphocyte egress during the
CE
very early (order of minutes) stages of exercise recovery are not known. The very early egress
kinetics could have important implications for studies that rely on standard phlebotomy to
assess lymphocyte mobilization and egress in response to an acute stress task. For instance, it
AC
may take several minutes to situate the subject following the task, gain intravenous access, and
draw the required volume of blood. The situation is further complicated by delays and
inconsistencies in sample collection time (even in the order of minutes) that may occur between
subjects and/or collection days. Individual and group variations in the rate of lymphocyte egress,
could also compromise those studies designed to make between and within subject
comparisons regarding leukocyte redeployment in response to an acute stress task. A potential
factor in determining the rate of leukocyte egress during the early stage of exercise recovery is
the time it takes to restore hemodynamic equilibrium. As heart rate returns to baseline after
exercise, so too does cardiac output, vagal tone, and blood pressure, thus allowing circulating
cells to adhere to their endothelial ligands and complete the process of diapedesis which
facilitates their translocation to the tissues [23].
ACCEPTED MANUSCRIPT
The aim of this study was to characterize the early (order of minutes) egress kinetics of blood
lymphocytes, monocytes, and their subtypes during the immediate recovery phase following a
single bout of steady state aerobic exercise. We hypothesized that lymphocytes and monocytes
would egress the blood compartment immediately upon exercise cessation, resulting in
circulating numbers falling below peak exercise values within just 5 minutes of passive recovery.
We further hypothesized that the most rapid egress rate within the lymphocytes would be
exhibited by NK-cells, followed by  T-cells, CD8+ T-cells, and then CD4+ T-cells. Finally, we
hypothesized that the rate of lymphocyte egress from the peripheral circulation would be
positively correlated with heart rate recovery following exercise cessation.
T
IP
1.2 Methods:
1.2.1 Participants:
CR
Eleven healthy and physically active individuals (7 males) between the ages of 26-38
participated in this study. All participants were considered ‘low’ risk for exercise testing as
US
defined by being asymptomatic for cardiovascular disease with less than two risk factors in
accordance with American College of Sports Medicine/American Heart Association
(ACSM/AHA) criteria [24]. Physical activity status was determined using the Jackson PA-R
questionnaire [25]. Participants reported regular engagement in vigorous and/or moderate
AN
intensity physical activity and did not report or exhibit any contraindications for participation in
our study as determined by our inclusion/exclusion criteria. Exclusion criteria included excessive
alcohol consumption, immunological impairments or diseases, and the taking of any
M
medications/supplements known to affect the immune system or cardiovascular system.

Additionally, all participants were required to be infection free for at least 6 weeks prior to their
ED
trial (confirmed by self report). Participants were asked to avoid strenuous activity 24 hours
before each visit. We obtained written informed consent from each participant, and the
Institutional Review Board (IRB) at the University of Houston granted approval for the study.
Descriptive statistics for the participants are summarized in (Table 1).
PT
1.2.2 Experimental protocol:

CE
Participants visited the lab on two separate occasions at the same time of day with no less than
24 hours and no more than 5 days between visits. On visit 1, participants completed an
incremental submaximal cycling protocol on a stationary cycle ergometer (Velotron® LABs
model Racermate Inc. Seattle, WA). The initial power output of 75W-100W was increased by
AC
10W every three minutes until a steady state heart rate equivalent to 80% of age predicted
maximum heart rate was attained, using the equation [191.5 – (.007*age2)]*0.80 [26]. Time to
target heart rate ranged between 10-20 minutes. The relationships between heart rate and
power output were plotted for each individual, and peak power was extrapolated from the curve
to coincide with age-predicted maximum heart rate. VO2max was estimated from the submaximal
exercise test using the YMCA prediction equations [27]. On visit 2, an indwelling intravenous
(IV) catheter was placed in a superficial forearm vein prior to exercise. Following 5 minutes of
seated rest on the bike, a baseline blood sample was drawn into a 5ml vacuum tube spray
coated with EDTA. Participants then completed a 30-minute bout of steady state cycling
exercise. Each participant was asked to maintain the power output corresponding to 80% of
their age-predicted maximum heart rate as determined from the submaximal cycling test
ACCEPTED MANUSCRIPT
performed during Visit 1. Participants provided ratings of perceived exertion (RPE) every five
minutes during exercise using the Borg scale [28], and power adjustments were made during
the test only if the subject intimated that they would not be able to maintain the required power
output for the entire 30-minute duration. Further blood samples were collected during exercise
(E) at +15 and +30 minutes. Participants were asked to continue pedaling against the resistance
of the cycle ergometer while the blood samples were being drawn. After collecting the blood
sample at E+30, participants were asked to stop pedaling and remain seated on the bike for 10
minutes of passive recovery (R). Further blood samples were drawn at exactly +1, +2, +3, +4,
+5 and +10 minutes following exercise cessation. The catheter was flushed with 3ml of non-
heparinized isotonic saline to maintain patency as required. Whenever the catheter was flushed,
T
a 3ml volume was collected from the IV catheter and discarded prior to collecting the blood
IP
sample. We recorded heart rate continuously using short-range telemetry (Polar RS300X
Electro Oy, Kempele, Finland) during the exercise bout and the recovery period. The exercise
CR
performance measures collected during the test are shown in Table 1.
1.2.3 Complete Blood Counts, immunofluorescence and Flow Cytometry:
Complete blood counts for leukocytes, lymphocytes, monocytes and granulocytes were
US
determined in duplicate using a clinical grade Hematology Analyzer (Mindray BC 3200,
Shenzhen, China). Direct immunofluorescence assays were performed to identify the numbers
and proportions for the following: CD3+, CD3+/CD4+, CD3+/CD8+ T-Cells, CD3+/CD4-/CD8- γδ
AN
T-Cells, CD3-/CD56+ NK-Cells, and for CD14++/CD16- (classical monocytes), CD14++/CD16+
(intermediate monocytes), and CD14+/CD16++ (pro-inflammatory monocytes). Briefly, 50μl of
whole blood was labeled with two separate antibody cocktails: (i) FITC-conjugated anti-CD56
M
(IgG1, TULY56), PE-conjugated anti-CD4 (IgG2b, OKT4), PerCy5-conjugated anti-CD8 (IgG1,

RPA-T8) and APC-conjugated anti-CD3 (IgG2a, OKT3); (ii) FITC-conjugated anti-CD14 (IgG1,
61D3), PE-conjugated anti-CD16 (IgG1, CB16), PerCy5-conjugated anti-CD20 (IgG2b, 2H7)
ED
and APC-conjugated anti-CD66 (IgG2a, CD66a-B1.1). All antibodies were mouse anti-human
monoclonal and purchased from eBioscience (San Diego, CA, USA). Single color control tubes
were used to adjust for spectral overlap during analysis by 4 color flow cytometry, using either
PT
an Accuri C6 flow cytometer (BD Accuri, Ann Arbor, MI) with (CFlow® software V2) or
MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) with (MACSQuantify™
Version 2.8). The total number of lymphocyte and monocyte subtypes in peripheral blood was
CE
determined by multiplying the percentage of all gated cells (lymphocytes or monocytes)

expressing the surface marker combinations of interest by the total blood lymphocyte or blood
monocyte counts (as determined by the automated hematology analyzer).
AC
1.2.4 Statistical Analysis

Prior to analysis, the data were screened to ensure that all test assumptions had been met.
Normality was confirmed using histograms and Fisher’s skewness and kurtosis coefficient, as
well as descriptive statistics and normal Q-Q plots. Mauchly’s test indicated that the assumption
of spherecity was violated for each of the cell types evaluated which led us to use the
Greenhouse–Geisser correction test. Repeated-measures ANOVA was utilized to assess the
change in leukocytes and lymphocytes over time as a main effect. Separate models were
developed to evaluate all leukocyte and lymphocyte subsets present in the peripheral blood
over time (resting, E+15, E+30, R+1, R+2, R+3, R+4, R+5 and R+10), as well as to assess the
percentage difference from E+30 values at all recovery time points (R+1 to R+10). Bonferroni
corrected, post hoc comparisons were used to determine which exercise recovery time points
ACCEPTED MANUSCRIPT
were significantly different from peak exercise (E+30). Bivariate correlation was used to assess
the relationship between the percent of E+30 heart rate and the percent of E+30 cell counts at
all exercise recovery time points (R+1 to R+10) for all cell types analyzed. SPSS version 22
(IBM; Armonk, NY, USA) was used for statistical analyses with significance indicated as (p<.05).
1.3 Results:
1.3.1 The egress of leukocytes and their subtypes is evident within just 1-3 minutes of exercise
recovery
There was a statistically significant main effect of time for leukocytes, lymphocytes, monocytes,
T
and granulocytes, (Table 2). Total leukocytes, lymphocytes, monocytes and granulocytes were
IP
mobilized in response to exercise, increasing above resting values at E+15 and reaching peak
concentrations in blood at E+30. Immediately upon cessation of exercise, cell numbers began to
decline; compared to E+30, total leukocytes were significantly lower by R+1, while lymphocyte
CR
and granulocyte numbers declined to statistically significant values by R+2 (p<.05). Monocyte
numbers were significantly lower than E+30 values by R+3 (p<.05) (Table 2). The R+3 and
R+10 total monocyte numbers were not statistically different to rest (p>0.99). Individual data for
US
these cell types expressed as a percent of the peak exercise cell counts at E+30 are shown in
Figure 1.
Lymphocyte and lymphocyte subsets were mobilized in response to exercise and then
AN
decreased from their E+30 values upon completion of exercise. A statistically significant main
effect of time was found for CD3+T-Cells, CD4+ T-Cells, CD8+ T-Cells, γδ T-Cells, and NK-
Cells (Table 2). Circulating cell counts became significantly different from E+30 at R+2 for NK-
M
Cells (p=.003), CD8+ T-Cells (p=.012), and CD3+ T-Cells (p=.025). CD4+ cell counts became
significantly different from E+30 at R+3, (p=.009), (Table 2). There was no statistical difference
ED
between absolute cell counts when comparing rest to R+4, R+5 or R+10 for CD3+, CD4+ and
CD8+ T-Cells, (p>0.99). Individual data for these cell types expressed as a percent of the peak
exercise cell counts at E+30 in Figure 2, with the NK-cell data being presented in Figure 3.
PT
Monocyte subpopulations increased during exercise and egressed immediately upon cessation
of exercise, with a statistically significant main effect of time for classical monocytes,
intermediate monocytes, and pro-inflammatory monocytes (Table 2). Classical monocytes
CE
showed a significant and consistent absolute cell number decline from E+30 (p<.009), beginning
at R+3 minutes and continuing through all measured post exercise time points. The egress for
pro-inflammatory monocytes showed a transient statistical significance at R+3, (Table 2).
AC
Additionally, for all the subpopulations of monocytes, there was statistically no difference when
comparing resting to recovery time points beyond R+3, (p>0.99). Individual data for these cell
types expressed as a percent of the peak exercise cell counts at E+30 are shown in Figure 3.
1.3.2 Leukocyte subtypes egress peripheral blood at different rates during exercise recovery
The relative rate of egress as a percentage of their respective peak values among the leukocyte
and lymphocyte subtypes was compared. The relative rate of egress from E+30 was variable
and contingent upon cell type, finding a significant main effect for time (F3.6, 143.26 = 129.61,
p<0.001) and a time x cell type interaction effect (F10.7, 143.26 = 3.99, p<0.001).Though the relative
egress from E+30 was statistically significant for all leukocyte and leukocyte subset counts at
R+1, (all, p<.05), monocytes showed the most rapid egress rate with a -14% change from E+30
at R+1. This was followed by -26% for R+2, a consistent -39-40% for R+3, R+4, and R+5, and
ACCEPTED MANUSCRIPT
finally -46% at R+10. Furthermore, the percent decline for monocytes was significantly greater
than leukocytes (p<.001), lymphocytes (p<.002), and granulocytes (p<.001), starting at R+2,
(Figure 4, top).
The rates of egress from E+30 values for lymphocytes and subsets again vary among the
different cell types, with a significant main effect of both time (F3.5, 175 = 93.6, p<0.001) and a
time x cell type interaction effect (F14, 175 = 3.24, p<0.001) being found. Though all lymphocyte
subset counts were significantly different from E+30 as early as R+1 (p<.05), NK-cells exhibited
a more rapid egress compared to the other lymphocyte subsets. NK-cell counts decreased -
10% from E+30 at R+1, -21% at R+2, -34% at R+3, -45% at R+4 and R+5 and -59% by R+10.
T
Additionally, the relative egress of NK-Cells was significantly greater from CD3+ T-Cells
(p<.001), CD4+ T-Cells (p<.001), CD8+ T-Cells (p<.004), and γδ T-Cells (p<.05), for all recovery
IP
time points starting at R+3, (Figure 4, bottom). NK-cells are followed, in order of egress rate,
by CD8+, γδ, and CD4+ T-cells.
CR
1.3.3 Lymphocyte, but not monocyte, egress correlates with decreasing heart rate during
passive recovery from exercise.
US
To determine if the rate of cell egress correlates with recovery heart rate, cell numbers in blood
during the recovery phase of exercise were expressed as a percentage of the E+30 cell count
and correlated with recovery heart rate as a percentage of the E+30 heart rate. The rate of
AN
egress for CD3+, CD4+, and CD8+ T-Cells, as well as NK-Cells all correlated (r >.5)
significantly with the decrease in heart rate for the first 2 minutes into passive recovery.
Recovery heart rate did not correlate with the egress of total lymphocytes, monocytes or
M
monocyte subtypes (Table 3).

1.4 Discussion
ED
We characterized for the first time the immediate (order of minutes) post-exercise kinetics of
leukocyte, lymphocyte, and monocyte subsets as they egressed the peripheral circulation upon
cessation of a single bout of steady state exercise. We determined that this egress occurs
PT
almost immediately, with the absolute numbers of all circulating leukocyte and leukocyte
subtypes falling below peak exercise values within just two minutes of passive recovery.
Further, total monocytes were not different to resting values after just 3 minutes of recovery,
CE
while CD3+, CD4+ and CD8+ T-cells were comparable to resting values after just 4, 5 and 10
minutes of passive recovery, respectively. These findings illustrate the rapid rate by which
leukocytes are redeployed to and from the blood compartment in response to an acute stress
AC
task. This also underscores the importance of timing when collecting blood samples after
exercise for the enumeration of circulating leukocytes and their subtypes, particularly when the
purpose is to capture peak cell mobilization during the stress task. We also found that the rate
of egress was associated with heart rate recovery for lymphocytes, indicating that the
restoration of hemodynamic equilibrium could be a key factor governing the rate of lymphocyte
egress from the blood compartment during the initial stages of exercise recovery.
Effective trafficking of immune cells through the lymphoid and peripheral tissues via the
circulation is essential to both immunosurveillance and maintaining host immunity [29]. We
contend that the speed of this exchange is also highly important because the lymphocyte and
monocyte subtypes that are preferentially mobilized by exercise exhibit greater effector function
(i.e. cytotoxicity), antigen experience and differentiation [7–11]. We further purport that the
ACCEPTED MANUSCRIPT
capacity of the host to rapidly redeploy leukocytes between the blood and tissues in response to
acute stress or exercise provides a global indication of immune system competence, as well as
the likelihood that an individual will resolve challenges to the immune system during or
immediately after a short-term stress task, [30]. Thus, for a given biological stress response
under controlled conditions (i.e. dose-controlled epinephrine infusion, individualized intensity
controlled exercise), a larger and swifter exchange of leukocytes between the blood and tissues
would be indicative of enhanced immunosurveillance and better global immunity, whereas a
smaller and/or slower rate of exchange might be indicative of compromised immunosurveillance
and an impaired ability to resolve immunological challenges. Although this remains to be
determined empirically, establishing the magnitude and rate of lymphocyte and monocyte
T
egress during exercise recovery may help stratify individuals with robust and compromised
IP
immune responses to stress. This could be useful, for example, to identify athletes at an
increased risk of infection or ‘burnout’ in response to overreaching or high volume/intensity
CR
training. Indeed, it has been shown that lymphocyte redeployment is reduced in highly trained
athletes at a given intensity of exercise following a period of functional overreaching [31]. Witard
et al. showed that just one week of intensified cycling training impaired the redistribution
(mobilization and egress) of CD8+ T-cells in response to a single bout of exercise, implying that
US
high intensity exercise training may impair immune surveillance in athletes [31].
Pro-inflammatory (non-classical) CD16+ monocytes and NK-cells egress the peripheral
AN
circulation more rapidly than other leukocytes. This is likely due to the importance of their
specific and immediate roles in host defense as part of the innate immune response.
Additionally, NK-cell egress may be related to the direct influence of macrophage derived
cytokines [32]. Both NK-cells and monocytes have higher relative densities of β2-adrenergic
M
receptors on their cell surface in comparison to the other leukocytes and lymphocytes [33,34].
This perhaps governs their rapid mobilization kinetics, [35], however their egress is more likely
ED
influenced by exercise induced increases in both glucocorticoids [22,36,37,42] and a myriad of

inflammatory chemokines/cytokines [38–41]. For instance, in a detailed temporal analysis of
lymphocyte trafficking using blood and saliva samples collected every 15-minutes in a 8h
PT
period, Trifonova et al. showed that diurnal changes in lymphocyte trafficking, including NK-
cells, was strongly linked to salivary cortisol, [42]. Monocytes may egress the blood
compartment under the influence of IL-6, which is known to be secreted at relatively high levels
CE
during exercise by contracting skeletal muscle even in the absence of muscle damage [43,44].
Monocytes are highly responsive to IL-6 as it stimulates their differentiation into macrophages
[45], where they phagocytize foreign substances at the tissues and aid in tissue repair. Though
this differentiation is not likely to occur in the 2 minutes of passive recovery, the monocytes
AC
might still home to areas where IL-6 is being produced. IL-6 also drives post-exercise NK-cell
homing [46], and due to NK-cell tissue homing receptor densities they predominantly home to
lung, spleen and muscle [47], where they can respond rapidly to opportunistic invading
pathogens or reactivating viruses. It is likely that a combination of catecholamines,
glucocorticoids and cytokines facilitate the rapid egress of discrete leukocyte subpopulations
from the peripheral blood compartment after exercise allowing their homing to areas where
reparative and sentinel action is required.
We also found a positive correlation between heart rate recovery and lymphocyte egress during
the first two minutes of exercise recovery. At this point, the heart rate had dropped ~28% from
peak values and, because heart rate recovery from exercise is more rapid among trained
individuals compared to those who are untrained [48], variability in egress rate is likely due to
ACCEPTED MANUSCRIPT
the broad range of aerobic fitness levels in this cohort of participants, (estimated VO2max 29 -
65.5 mlkg-1min-1). Lymphocyte egress from peripheral blood involves the physical process of
diapedesis. This requires the migrating leukocytes to first adhere to the vascular endothelium,
initially through tethering and rolling adhesion that is mediated by selectins and endothelial
ligands such as CD34 [23]. This is followed by activation and firm adhesion, which requires
ligation of integrins with stronger adhesion molecules on the endothelium such as ICAM-1,
allowing the leukocyte to attach firmly against the forces of blood flow for subsequent migration
through adjacent endothelial cells toward the tissues [23]. We purported, therefore, that a swifter
restoration towards hemodynamic equilibrium (i.e. lowering of cardiac output, blood flow and
blood pressure) during exercise recovery would allow for diapedesis and a more rapid
T
extravasation of the leukocytes previously mobilized by exercise to take place. As such, our
IP
finding that lymphocyte egress correlated positively with heart rate recovery (used here as a
global indicator of hemodynamic restoration) during the first few minutes of exercise cessation
CR
was expected. This does indicate, however, that the rate of lymphocyte egress during the early
stages of exercise recovery is likely to be influenced by aerobic fitness, and a future study
comparing lymphocyte and monocyte egress rates between trained and untrained individuals
after intensity controlled exercise would be illuminating. It will also be important for future
US
studies to consider the many other factors that could also play a role in the rate of leukocyte
egress after exercise such as age, sex, catecholamines, glucocorticoids, cytokines, infection
history, leukocyte composition, adrenergic/glucocorticoid receptor density/sensitivity and
AN
cardiovascular responses, and also to characterize, in great detail, leukocyte trafficking beyond
10-minutes of recovery.
These findings also have practical implications for research studies in exercise and stress
M
immunology. Many studies in this field are concerned with the effects of acute bouts of physical
(i.e. exercise) or psychological stress on leukocyte redistribution to and from the blood
ED
compartment. Oftentimes, the post-stress blood draw is collected ‘immediately’ after the stress
task under the assumption that this will represent peak cell numbers mobilized during the stress
task. While there is a clear lack of consistency and specificity when detailing the time-course of
PT
when blood samples are collected relative to the onset and cessation of acute stress [49], some
exercise studies are specific in detailing the time elapsed after exercise when the ‘post-exercise’
blood sample was actually drawn, which can sometimes be as long as 5-minutes [50–52]. As
CE
standard venipuncture is most often used for the collection of ‘post-exercise’ blood samples, it
can be assumed that these blood draws actually occur during the first few minutes of exercise
recovery. Our findings would indicate that blood samples collected, even just a few minutes
AC
after exercise cessation, are suboptimal for identifying peak cell mobilization during exercise.
Moreover, even if the amount of time elapsed following exercise cessation is defined (i.e. 5-
minutes) and consistent prior to blood collection, it is still likely that differences in lymphocyte
and monocyte egress rates among subjects, possibly due to differences in fitness levels, would
remain an important confounder. It is clear from our findings that, in order to accurately capture
peak cell mobilization during exercise, future studies should use indwelling intravenous
catheters over standard phlebotomy. This would allow samples to be drawn while the subject is
still performing the exercise bout at the prescribed intensity, ensuring that the window to capture
peak cell mobilization is not missed.
1.5 Conclusion
ACCEPTED MANUSCRIPT
Lymphocytes and monocytes begin to egress the peripheral blood compartment immediately
upon cessation of a single bout of steady state exercise. Significant numbers of lymphocytes
and monocytes extravasate the bloodstream within as little as 3 minutes of passive recovery,
with those monocyte and lymphocyte subtypes known to have phenotypes associated with
increased effector function and tissue migration (i.e. pro-inflammatory monocytes, NK-cells,
CD8+ T-cells) exhibiting the most rapid egress kinetics. The rate of monocyte and lymphocyte
egress may be dependent on how quickly hemodynamic perturbations are restored following an
acute bout of exercise, indicating that individual fitness levels are likely to play a role in
determining how quickly certain leukocyte subtypes can exit the blood compartment and migrate
toward the tissue during exercise recovery.
T
Acknowledgements
IP
We would like to thank Rod Azadan, Forrest Baker and Rachel Graff for their technical support.
CR
This work was supported by NASA Grants NNJ10ZSA003N, NNJ14ZSA001N-FLAGSHIP and
NNJ14ZSA001N-MIXEDTOPICS and NIH Grant R21 CA197527-01A1.
Conflicts of Interest
US
None AN
M
ED
PT
CE
AC
ACCEPTED MANUSCRIPT
References:
[1] R.J. Simpson, The effects of exercise on blood leukocyte numbers, in: M. Gleeson, N.
Bishop, N. Walsh (Eds.), Exerc. Immunol., Routledge: Oxford, UK, 2013: pp. 64–105.
[2] N. Walsh, M.M. Gleeson, R. Shephard, Position statement part one: immune function and
exercise, Exerc. Immunol. Rev. 17 (2011) 6–63. https://dspace.lboro.ac.uk/dspace-
jspui/handle/2134/10584%5Cnhttps://dspace.lboro.ac.uk/xmlui/handle/2134/10584.
[3] P.J. Robson, a K. Blannin, N.P. Walsh, L.M. Castell, M. Gleeson, Effects of exercise
intensity, duration and recovery on in vitro neutrophil function in male athletes., Int. J.
Sports Med. 20 (1999) 128–135. doi:10.1055/s-2007-971106.
T
IP
[4] R.J. Simpson, B.K. McFarlin, C. McSporran, G. Spielmann, B. ó Hartaigh, K. Guy, Toll-
like receptor expression on classic and pro-inflammatory blood monocytes after acute
exercise in humans, Brain. Behav. Immun. 23 (2009) 232–239.
CR
doi:10.1016/j.bbi.2008.09.013.
[5] P.N. Shek, B.H. Sabiston, A. Buguet, M.W. Radomski, Strenuous exercise and
immunological changes: A multiple-time-point analysis of leukocyte subsets, CD4/CD8
US
ratio, immunoglobulin production and NK cell response, Int. J. Sports Med. 16 (1995)
466–474. doi:10.1055/s-2007-973039.
[6] H. Gabriel, L. Schwarz, P. Born, W. Kindermann, Differential mobilization of leucocyte
AN
and lymphocyte subpopulations into the circulation during endurance exercise, Eur. J.
Appl. Physiol. Occup. Physiol. 65 (1992) 529–534. doi:10.1007/BF00602360.
M
[7] L.H. Anane, K.M. Edwards, V.E. Burns, M.T. Drayson, N.E. Riddell, J.J.C.S.V. van
Zanten, G.R. Wallace, P.J. Mills, J.A. Bosch, Mobilization of gammadelta T lymphocytes
in response to psychological stress, exercise, and beta-agonist infusion., Brain. Behav.
ED
Immun. 23 (2009) 823–9. doi:10.1016/j.bbi.2009.03.003.

[8] A.B. Bigley, T.W. Lowder, G. Spielmann, J.L. Rector, H. Pircher, J.A. Woods, R.J.
Simpson, NK-cells have an impaired response to acute exercise and a lower expression
PT
of the inhibitory receptors KLRG1 and CD158a in humans with latent cytomegalovirus
infection, Brain. Behav. Immun. 26 (2012) 177–186. doi:10.1016/j.bbi.2011.09.004.
[9] J.P. Campbell, N.E. Riddell, V.E. Burns, M. Turner, J.J.C.S.V. van Zanten, M.T. Drayson,
CE
J.A. Bosch, Acute exercise mobilises CD8+ T lymphocytes exhibiting an effector-memory

phenotype, Brain. Behav. Immun. 23 (2009) 767–775. doi:10.1016/j.bbi.2009.02.011.
AC
[10] B.W. Timmons, T. Cieslak, Human natural killer cell subsets and acute exercise: A brief
review, Exerc. Immunol. Rev. 14 (2008) 8–23.
[11] E.C. LaVoy, J.A. Bosch, T.W. Lowder, R.J. Simpson, Acute aerobic exercise in humans
increases cytokine expression in CD27- but not CD27+ CD8+ T-cells, Brain. Behav.
Immun. 27 (2013) 54–62. doi:10.1016/j.bbi.2012.09.006.
[12] F.S. Dhabhar, Enhancing versus suppressive effects of stress on immune function:
Implications for immunoprotection and immunopathology, Neuroimmunomodulation. 16
(2009) 300–317. doi:10.1159/000216188.
[13] M. Okutsu, K. Ishii, K.J. Niu, R. Nagatomi, Cortisol-induced CXCR4 augmentation
mobilizes T lymphocytes after acute physical stress., Am. J. Physiol. Regul. Integr.
Comp. Physiol. 288 (2005) R591-9. doi:10.1152/ajpregu.00438.2004.
ACCEPTED MANUSCRIPT
[14] R.J. Simpson, Aging, persistent viral infections, and immunosenescence: can exercise
“make space”?, Exerc. Sport Sci. Rev. 39 (2011) 23–33.
doi:10.1097/JES.0b013e318201f39d.
[15] R.J. Simpson, G.D. Florida-James, C. Cosgrove, G.P. Whyte, S. Macrae, H. Pircher, K.
Guy, High-intensity exercise elicits the mobilization of senescent T lymphocytes into the
peripheral blood compartment in human subjects., J. Appl. Physiol. 103 (2007) 396–401.
doi:10.1152/japplphysiol.00007.2007.
[16] R.J. Simpson, C. Cosgrove, L.A. Ingram, G.D. Florida-James, G.P. Whyte, H. Pircher, K.
Guy, Senescent T-lymphocytes are mobilised into the peripheral blood compartment in
T
young and older humans after exhaustive exercise, Brain. Behav. Immun. 22 (2008) 544–
551. doi:10.1016/j.bbi.2007.11.002.
IP
[17] F.S. Dhabhar, W.B. Malarkey, E. Neri, B.S. McEwen, Stress-induced redistribution of
CR
immune cells-From barracks to boulevards to battlefields: A tale of three hormones - Curt
Richter Award Winner, Psychoneuroendocrinology. 37 (2012) 1345–1368.
doi:10.1016/j.psyneuen.2012.05.008.
US
[18] R.L. Berkow, R.W. Dodson, Functional analysis of the marginating pool of human
polymorphonuclear leukocytes, Am. J. Hematol. 24 (1987) 47–54.
doi:10.1002/ajh.2830240107.
AN
[19] J.C. Hogg, C.M. Doerschuk, Leukocyte Traffic in the Lung, Annu. Rev. Physiol. 57 (1995)
97–114. doi:10.1146/annurev.ph.57.030195.000525.
[20] H.B. Nielsen, N.H. Secher, J.H. Kristensen, N.J. Christensen, K. Espersen, B.K.
M
Pedersen, Splenectomy impairs lymphocytosis during maximal exercise., Am. J. Physiol.

272 (1997) R1847–R1852. doi:10.1097/00005768-199605001-00550.
ED
[21] S. Dimitrov, T. Lange, J. Born, Selective mobilization of cytotoxic leukocytes by

epinephrine., J. Immunol. 184 (2010) 503–511. doi:10.4049/jimmunol.0902189.
[22] M. Okutsu, K. Suzuki, T. Ishijima, J. Peake, M. Higuchi, The effects of acute exercise-
PT
induced cortisol on CCR2 expression on human monocytes, Brain. Behav. Immun. 22

(2008) 1066–1071. doi:10.1016/j.bbi.2008.03.006.
[23] W.A. Muller, Getting Leukocytes to the Site of Inflammation, Vet. Pathol. 50 (2013) 7–22.
CE
doi:10.1177/0300985812469883.
[24] L.S. Pescatello, ACSM Guidelines for Exercise Testing and Prescription, 9th Edition,
2014.
AC
[25] A.S. Jackson, S.N. Blair, M.T. Mahar, L.T. Wier, R.M. Ross, J.E. Stuteville, Prediction of
functional aerobic capacity without exercise testing., Med. Sci. Sports Exerc. 22 (1990)
863–70. doi:10.1249/00005768-199012000-00021.
[26] R.L. Gellish, B.R. Goslin, R.E. Olson, A. McDonald, G.D. Russi, V.K. Moudgil,
Longitudinal modeling of the relationship between age and maximal heart rate, Med Sci
Sport. Exerc. 39 (2007) 822–829. doi:10.1097/mss.0b013e31803349c6.
[27] L. Golding, W. Sinning, Y ’ s Way to Physical Fitness: The Complete Guide to Fitness
Testing and Instruction: YMCA of the USA, Champaign, Hum. Kinet. Publ. (1989).
[28] G. Borg, Perceived exertion as an indicator of somatic stress., Scand. J. Rehabil. Med. 2
(1970) 92–98. doi:S/N.
ACCEPTED MANUSCRIPT
[29] U.H. von Andrian, C.R. Mackay, T-Cell Function and Migration — Two Sides of the Same
Coin, N. Engl. J. Med. 343 (2000) 1020–1034. doi:10.1056/NEJM200010053431407.
[30] J.P. Campbell, J.E. Turner, Debunking the Myth of Exercise-Induced Immune
Suppression: Redefining the Impact of Exercise on Immunological Health Across the
Lifespan, Front. Immunol. 9 (2018) 648. doi:10.3389/fimmu.2018.00648.
[31] O.C. Witard, J.E. Turner, S.R. Jackman, K.D. Tipton, A.E. Jeukendrup, A.K. Kies, J.A.
Bosch, High-intensity training reduces CD8+ T-cell redistribution in response to exercise,
Med. Sci. Sports Exerc. 44 (2012) 1689–1697. doi:10.1249/MSS.0b013e318257d2db.
[32] M.A. Caligiuri, Human natural killer cells, Blood. 112 (2008) 461–469. doi:10.1182/blood-
T
2007-09-077438.
IP
[33] R. Landmann, Beta-adrenergic receptors in human leukocyte subpopulations., Eur. J.
Clin. Invest. 22 Suppl 1 (1992) 30–6.
CR
[34] R. Graff, R.A. Bond, H. Kunz, N.H. Agha, F.L. Baker, R. Azadan, B. V Rooney, P.L.
Mylabathula, E.C. LaVoy, M. Laughlin, S.C. Gilchrist, C.M. Bollard, R.J. Simpson, The
relative contribution of beta2-adrenergic receptor signaling to the mobilization of
US
lymphocyte and monocyte subsets in response to acute dynamic exercise in humans,
Brain. Behav. Immun. 66 (2017) e30. doi:https://doi.org/10.1016/j.bbi.2017.07.112.
[35] K. Krüger, A. Lechtermann, M. Fobker, K. Völker, F.C. Mooren, Exercise-induced
AN
redistribution of T lymphocytes is regulated by adrenergic mechanisms, Brain. Behav.
Immun. 22 (2008) 324–338. doi:10.1016/j.bbi.2007.08.008.
[36] J.M. Peake, O. Neubauer, N.P. Walsh, R.J. Simpson, Recovery of the immune system
M
after exercise, J. Appl. Physiol. 122 (2017) 1077 LP-1087.

http://jap.physiology.org/content/122/5/1077.abstract.
ED
[37] M. Okutsu, K. Ishii, K. Niu, R. Nagatomi, Cortisol is not the primary mediator for
augmented CXCR4 expression on natural killer cells after acute exercise., J. Appl.
Physiol. 117 (2014) 199–204. doi:10.1152/japplphysiol.00176.2014.
PT
[38] B.K. Pedersen, Exercise and cytokines, Immunol. Cell Biol. 78 (2000) 532–535.
doi:10.1046/j.1440-1711.2000.00962.x.
CE
[39] F. Zaldivar, J. Wang-Rodriguez, D. Nemet, C. Schwindt, P. Galassetti, P.J. Mills, L.D.

Wilson, D.M. Cooper, Constitutive pro- and anti-inflammatory cytokine and growth factor
response to exercise in leukocytes, J Appl Physiol. 100 (2006) 1124–1133.
doi:00562.2005 [pii]\r10.1152/japplphysiol.00562.2005.
AC
[40] J. Peake, P. Della Gatta, K. Suzuki, D. Nieman, Cytokine expression and secretion by
skeletal muscle cells: regulatory mechanisms and exercise effects., Exerc. Immunol. Rev.
21 (2015) 8–25. doi:10.1113/EXPPHYSIOL.2012.068189.
[41] K. Ostrowski, T. Rohde, S. Asp, P. Schjerling, B.K. Pedersen, Chemokines are elevated
in plasma after strenuous exercise in humans., Eur. J. Appl. Physiol. 84 (2001) 244–245.
doi:10.1007/s004210170012.
[42] S.T. Trifonova, J. Zimmer, J.D. Turner, C.P. Muller, Diurnal redistribution of human
lymphocytes and their temporal associations with salivary cortisol, Chronobiol. Int. 30
(2013) 669–681. doi:10.3109/07420528.2013.775654.
[43] A. Steensberg, M.A. Febbraio, T. Osada, P. Schjerling, G. Van Hall, B. Saltin, B.K.
ACCEPTED MANUSCRIPT
Pedersen, Interleukin-6 production in contracting human skeletal muscle is influenced by

pre-exercise muscle glycogen content, J. Physiol. 537 (2001) 633–639.
doi:10.1111/j.1469-7793.2001.00633.x.
[44] B.K. Pedersen, A. Steensberg, P. Schjerling, Muscle-derived interleukin-6: Possible
biological effects, J. Physiol. 536 (2001) 329–337. doi:10.1111/j.1469-
7793.2001.0329c.xd.
[45] P. Chomarat, J. Banchereau, J. Davoust, a K. Palucka, IL-6 switches the differentiation
of monocytes from dendritic cells to macrophages., Nat. Immunol. 1 (2000) 510–514.
doi:10.1038/82763.
T
[46] L. Pedersen, M. Idorn, G.H. Olofsson, B. Lauenborg, I. Nookaew, R.H. Hansen, H.H.
IP
Johannesen, J.C. Becker, K.S. Pedersen, C. Dethlefsen, J. Nielsen, J. Gehl, B.K.
Pedersen, P. Thor Straten, P. Hojman, Voluntary running suppresses tumor growth
CR
through epinephrine- and IL-6-dependent NK cell mobilization and redistribution, Cell
Metab. 23 (2016) 554–562. doi:10.1016/j.cmet.2016.01.011.
[47] G.R. Adams, F.P. Zaldivar, D.M. Nance, E. Kodesh, S. Radom-Aizik, D.M. Cooper,
US
Exercise and leukocyte interchange among central circulation, lung, spleen, and muscle,
Brain. Behav. Immun. 25 (2011) 658–666. doi:10.1016/j.bbi.2011.01.002.
[48] O. Kwon, S. Park, Y.J. Kim, S.Y. Min, Y.R. Kim, G.B. Nam, K.J. Choi, Y.H. Kim, The
AN
exercise heart rate profile in master athletes compared to healthy controls, Clin. Physiol.
Funct. Imaging. 36 (2016) 286–292. doi:10.1111/cpf.12226.
[49] A.M. Szlezak, S.L. Szlezak, J. Keane, L. Tajouri, C. Minahan, Establishing a dose-
M
response relationship between acute resistance-exercise and the immune system:

Protocol for a systematic review, Immunol. Lett. 180 (2016) 54–65.
doi:10.1016/j.imlet.2016.10.010.
ED
[50] V.M. Natale, I.K. Brenner, A.I. Moldoveanu, P. Vasiliou, P. Shek, R.J. Shephard, Effects
of three different types of exercise on blood leukocyte count during and following
exercise, Sao Paulo Med. J. 121 (2003) 9–14. doi:S1516-31802003000100003 [pii].
PT
[51] B.A. Risøy, T. Raastad, J. Hallén, K.T. Lappegård, K. Baeverfjord, A. Kravdal, E.M.
Siebke, H.B. Benestad, Delayed leukocytosis after hard strength and endurance
CE
exercise: aspects of regulatory mechanisms., BMC Physiol. 3 (2003) 14.

doi:10.1186/1472-6793-3-14.
[52] J.P. Morgado, C.P. Monteiro, C.N. Matias, F. Alves, P. Pessoa, J. Reis, F. Martins, T.
AC
Seixas, M.J. Laires, Sex-based effects on immune changes induced by a maximal

incremental exercise test in well-trained swimmers, J. Sport. Sci. Med. 13 (2014) 708–
714.
ACCEPTED MANUSCRIPT
Figure 1. Leukocytes and leukocyte subtypes rapidly egress the peripheral circulation
immediately upon cessation of steady state exercise. Individual data shown as a percentage of
E+30 cell counts during exercise recovery (n=11). The mean of all participants is shown as a
bold solid line.
Figure 2. T-Cells rapidly egress the peripheral circulation immediately upon cessation of steady
state exercise. Individual data shown as a percentage of E+30 cell counts during exercise
recovery (n=11). The mean of all participants is shown as a bold solid line.
Figure 3. NK-cells and monocyte subtypes rapidly egress the peripheral circulation immediately
upon cessation of steady state exercise. Individual data shown as a percentage of E+30 cell
T
counts during exercise recovery (n=11). The mean of all participants is shown as a bold solid
IP
line.
Figure 4. The relative egress of leukocytes/leukocyte subtypes (top) and lymphocyte subtypes
CR
(bottom) from peripheral blood immediately upon cessation of steady state exercise. Values are
mean ± SE. * indicates statistically significant difference from E+30 for all cell types, p<.005, #
indicates statistically significant difference between monocytes (top) and all other cell types
US
(top), and between NK Cells all other cell types (bottom), p<0.05.
AN
M
ED
PT
CE
AC
ACCEPTED MANUSCRIPT
Table 1. Physical characteristics and exercise performance measures of the participants (n=11).
All exercise performance measures were averaged across the 30-minute bout.
Physical Characteristics Mean SD Range

Age (Years) 31.0 4.4 25-38
Height (cm) 173.2 10.1 156-185.4
Mass (kg) 72.1 7.8 56.3-82
Body Mass Index (kgm-2) 24.0 1.9 20-27
T
Maximum Heart Rate (bpm)1 184.6 1.9 181-187.1
IP
Physical activity rating (0-7)2 5.1 1.8 2.0-7
Estimated Peak Cycling Power (W) 225.6 59.6 100.3-288.3
CR
VO2max (mlkg-1min-1)3 46.2 10.5 29-65.5
Exercise Performance Measures
Cycling Power (W) 150.6 36.3 75-200
US
Cycling Power (% of peak cycling power) 67.7 6.9 60-77
Exercising Heart Rate (bpm) 152.9 11.3 126-167
Exercising Heart Rate (% of predicted max) 82.9 6.6 67-91
AN
4
Perceived Exertion (Borg's 6-20 Scale) 14.1 1.5 11.7-17
1
Maximum Heart Rate was estimated using the equation 191.5 – (.007*age2) [26].
M
2
Physical activity rating was determined on a 0-7 point scale: None-0, Minimal-1, Moderate-2-3,
Vigorous-4-7 [25].
ED
3
Maximal oxygen uptake (VO2max) was estimated from the submaximal exercise test [27].
4
Rating of perceived exertion (RPE) was determined using the Borg 6-20 scale [28].
PT
CE
AC
ACCEPTED MANUSCRIPT
Table 2. Absolute number of circulating cells (cells/μL) for each experimental time point, with
results from the repeated measure ANOVA, (n=11). Values are mean ± SD.
Rest E+15 E+30 R+1 R+2 R+3 R+4 R+5 R+10 F (df) p
5429 ± 8067 ± 8533 ± 8058 ± 7550 ± 7129 ± 6858 ± 6683 ± 6225 ± <0.0
Leukocytes 58.2 (3,30)
816 * 1277 1506 1380 * 1294 * 1247 * 1116 * 1192 * 1092 * 01
1679 ± 2746 ± 2946 ± 2554 ± 2395 ± 2316 ± 2225 ± 2129 ± 39.96 <0.0
Lymphocytes 2745 ± 566
205 * 519 663 484 * 519 * 502 * 452 * 513 * (3.1,31) 01
383 ± 545 ± 550 ± 533 ± 483 ± <0.0
Monocytes 783 ± 272 887 ± 360 766 ± 323 654 ± 278 18 (3,30)
153 * 249 * 194 * 217 * 186 * 01
3367 ± 4350 ± 4195 ± 3991 ± 3916 ± 3600 ±
T
4546 ± 4704 ± 4546 ± 44.16 (2.4, <0.0
Granulocytes
825 * 1115 1135 1228 1156 * 1099 * 1021 * 1086 * 853 * 24) 01
1259 ± 1209 ± 1115 ± 1140 ± 982 ±
IP
1291 ± 1427 ± 11.2 (2.2, <0.0
CD3+ T-cells 884 ± 354 1351 ± 676
679 745 679 * 670 * 637 * 627 * 518 * 22) 01
517 ± 627 ± 579 ± 591 ± 515 ± 9.29 <0.0
CD4/CD3+ T-cells 668 ± 330 709 ± 341 680 ± 330 636 ± 328
233 * 322 * 325 * 323 * 271 * (3.2,32) 01
CR
454 ± 432 ± 395 ± 410 ± 350 ± 11.61 <0.0
CD8+/CD3+ T-cells 295 ± 123 467 ± 275 531 ± 304 515 ± 292
267 * 269 * 251 * 250 * 209 * (2.2,22) 01
13.71 (1.7, <0.0
γδ T Cells 194 ± 161 394 ± 307 425 ± 268 407 ± 273 391 ± 235 339 ± 230 323 ± 200 313 ± 200 303 ± 224
17) 01
442 ± 413 ± 345 ± 290 ± 289 ± 215 ± 32.41 <0.0
NK-cells 139 ± 86 * *
523 ± 269 469 ± 245
* * * * *
US
226 245 230 196 169 138 (2,21) 01
295 ± 407 ± 386 ± 390 ± 348 ± 16.62 <0.0
Classical Monocytes 598 ± 228 652 ± 288 563 ± 260 489 ± 226
119 * 202 * 203 * 189 * 150 * (3.4,34) 01
Intermediate 6.71 <0.0
35 ± 27 58 ± 31 80 ± 67 71 ± 54 56 ± 38 48 ± 43 44 ± 35 46 ± 39 40 ± 30
Monocytes (2.7,27) 02
AN
Pro-Inflammatory <0.0
Monocytes
27 ± 17 52 ± 46 74 ± 53 57 ± 35 49 ± 33 38 ± 31 * 41 ± 25 43 ± 32 31 ± 20 5.61 (3,30)
03
* indicates statistically significant difference from Exercise Phase +30 minutes (E+30) in
M
accordance with post-hoc Bonferroni correction, p<.05.

ED
PT
CE
AC
ACCEPTED MANUSCRIPT
Table 3. Bivariate correlations of heart rate recovery and cell egress when expressed as a
percentage of E+30 values during the early stages of recovery following cessation of steady
state exercise (n=11)
Recovery Time Point

R+1 R+2 R+3 R+4 R+5 R+10
Lymphocytes 0.599 0.454 0.187 0.124 0.434 -0.071
T
* *
CD3+ T-Cells 0.623 0.609 0.572 0.504 0.538 0.521
IP
* *
CD4+ T-Cells 0.622 0.589 0.559 0.454 0.492 0.458
CR
* *
CD8+ T-Cells 0.614 0.615 0.538 0.479 0.535 0.494
*
NK Cells 0.585 0.570 0.434 0.426 0.476 0.397
US
γδ T-Cells 0.470 0.043 0.272 0.392 .674* 0.204
AN
Total Monocytes 0.121 0.207 -0.130 0.414 0.201 0.032
Granulocytes -0.028 0.133 0.195 0.432 0.208 0.137

M
Classical Monocytes -0.414 -0.555 0.276 -0.008 -0.134 -0.147

ED
Intermediate Monocytes -0.469 -0.27 0.057 -0.076 0.082 -0.560
Pro-Inflammatory Monocytes -0.367 -0.336 0.232 0.105 0.498 -0.420

PT
Numbers presented in the table are Pearson correlation coefficients (r). * indicates statistically
significant correlation between % of E+30 heartrate and % of E+30 cell counts at the specified
CE
time point during exercise recovery, p<.05.

AC
ACCEPTED MANUSCRIPT
“Core findings”
Highlights:
 Lymphocytes and monocytes egress blood immediately upon cessation of acute

exercise
 This cellular egress is rapid and evident within just 3 minutes of passive recovery
 Monocytes egress the peripheral circulation more rapidly than other leukocytes
 NK-Cells egress the peripheral circulation more rapidly than other lymphocytes
 The rate of lymphocyte egress correlates with recovering heart rate after exercise
T
IP
CR
US
AN
M
ED
PT
CE
AC
Figure 1
Figure 2
Figure 3
Figure 4
www.nature.com/scientificreports
OPEN Association between Leukocyte

Counts and Physical Fitness in Male
Military Members: The CHIEF Study
Pei-Shou Chung1, Kun-Zhe Tsai1, Yen-Po Lin2, Yu-Kai Lin3 & Gen-Min Lin1,4,5*
Low-grade inflammation, which is related to obesity and toxic substance use in young adults, may be
associated with poor physical fitness. We investigated the association between total leukocyte count
and physical fitness in a military cohort of 3,453 healthy young Taiwanese males aged 20–50 years
in a cross-sectional study in 2014. Low-grade inflammation was defined according to equally sized
quartiles of total leukocyte counts within the suggested normal limits (4.00–9.99 × 103/mm3). Aerobic
fitness was assessed by the time for a 3-kilometer run test, and anaerobic fitness was evaluated by the
numbers of sit-ups and push-ups performed in 2 minutes. Automatic monitoring systems were used to
verify the scores for all procedures. Multiple linear regression was utilized to identify the associations
among variables. When compared with the lowest counts (4.00–5.49 × 103/mm3), the second highest
(6.50–7.49 × 103/mm3) and highest normal leukocyte counts (7.50–9.99 × 103/mm3) were correlated
with longer times for a 3-kilometer run (β and 95% confidence intervals =4.93 (1.61, 8.25) and 4.65
(2.20, 7.10), respectively) and fewer numbers of push-ups performed in 2 minutes (β = −0.59 (−1.15,
−0.03) and −0.56 (−0.96, −0.17), respectively), after adjustments for age, service specialty, waist
circumference, body mass index, alcohol consumption, tobacco smoking, and physical activity.
However, the association with 2-minute sit-ups was null. Our study suggested an inverse association
between total leukocyte count and not only aerobic fitness but also parts of anaerobic fitness in young
males. The temporal association needs confirmation in longitudinal studies.
Low-grade inflammation usually refers to a mild upregulation of cytokine and leukocyte levels in the blood1,2, and
it is related to a more pronounced activation state of immune cells3. In the absence of a septic process and chronic
diseases, proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)−1β and IL-6
can be generated by adipocytes4,5 and stimulate hepatocytes to produce C-reactive protein6, which further induces
bone marrow to increase circulating blood leukocyte (white blood cells) concentrations7. Metabolic abnormal-
ities, including overweight, obesity, hyperuricemia and atherosclerosis, have been associated with low-grade
inflammation in the body7. In addition, exposure to toxic substances such as tobacco smoke and alcohol bever-
ages can activate innate immunity8, leading to systemic inflammation. Furthermore, mental stress, such as anxiety
and posttraumatic stress disorder, could increase high-sensitivity C-reactive protein and decrease adiponectin
concentrations, where adiponectin is known as a cardio-protective marker9,10.
Several previous population studies including diabetic patients11, those with metabolic syndrome12, or appar-
ently healthy middle- and old-aged individuals14–16 have revealed that the degree of low-grade inflammation
evaluated by total leucocyte counts and C-reactive protein concentrations was dose-dependently and directly
associated with body fatness but inversely associated with cardiorespiratory fitness, as assessed by the maximum
oxygen consumption during a treadmill exercise test. However, studies on the association between low-grade
inflammation and muscular strength (anaerobic fitness) are lacking, and few studies have examined the associa-
tion with cardiorespiratory fitness in young adults.
To our knowledge, systemic low-grade inflammation in young adults is mainly associated with great body
adiposity and habitual toxic substance use, and it is not easily evaluated in large population studies. Since physi-
cal fitness is highly related to the performance of exercises, we aimed to investigate the association of low-grade
1
Department of Medicine, Hualien Armed Forces General Hospital, Hualien, Taiwan. 2Department of Critical Care
Medicine, Taipei Tzu-Chi Hospital, New Taipei, Taiwan. 3Departments of Neurology and Internal Medicine, Tri-Service
General Hospital, National Defense Medical Center, Taipei, Taiwan. 4Internal Medicine, Tri-Service General Hospital,
National Defense Medical Center, Taipei, Taiwan. 5Department of Preventive Medicine, Northwestern University
Feinberg School of Medicine, Chicago, IL, 60611, USA. *email: farmer507@yahoo.com.tw
Scientific Reports | (2020) 10:6082 | https://doi.org/10.1038/s41598-020-63147-9 1

www.nature.com/scientificreports/ www.nature.com/scientificreports
inflammation with aerobic and anaerobic fitness in a large military cohort of healthy young male adults who
participated in annual exercise tests in eastern Taiwan.
Methods
Study population. The study subjects were obtained from a military population of 6,133 young adults, aged
18–50 years, from the cardiorespiratory fitness and hospitalization events in armed forces (CHIEF) study per-
formed in Taiwan during 201417. All participants carried out a health screening involving a physical examination,
chest roentgenography, blood and urine tests in the Hualien Armed Forces General Hospital, and a self-reported
questionnaire regarding the habits of cigarette smoking, alcohol intake, and betel nut chewing status (current vs.
former or never) and total physical activity per week over the past 6 months. Before the laboratory studies, each
participant’s temporal temperature and general condition were checked, and each patient was interviewed by a
physician. If there were any infection symptoms or signs along with a body temperature over >37 °C, the par-
ticipant’s next visit was scheduled after full recovery from the sickness was achieved. The inclusion criteria were
the participants completing at least one of the three exercise tests, including sit-ups for 2 minutes, push-ups for
2 minutes and a 3000-meter run, for military awards or rank promotions at the Military Physical Testing Center in
Hualien after the health screening in 2014. As a result, 4,080 participants were included, and the response rate was
approximately 66.5%. The detailed design of the CHIEF study has been reported previously17–24. The exclusion
criteria were females with an active menstrual cycle, which could influence all blood cell counts (n = 411), and
those with total leukocyte counts <4,000 × 103/mm3 (n = 36) or >10,000 × 103/mm3 (n = 50) or with evidence
for volume depletions such as recent blood loss, diarrhea and plasma blood urea nitrogen >20 mg/dL (n = 130).
These criteria yielded a final sample of 3,453 males for analysis.
Laboratory assessment. For each participant, body height and weight were assessed in a standing posi-
tion. Body mass index was defined as the ratio of body weight (kilogram) to the square of height (meter2).
Measurement of waist circumference was taken at the midpoint between the highest point of the iliac crests
and the lowest point of palpable ribs. Blood pressure (BP) was measured after resting for at least 25 minutes and
taken once from the right arm in a sitting position with an automated FT-201 BP monitor (Parama-Tech Co Ltd,
Fukuoka, Japan). The metabolic panel of blood biochemical tests for serum triglycerides, fasting plasma glucose,
low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and total cholesterol were measured on
an AU640 autoanalyzer (Olympus, Kobe, Japan). Routine blood tests, including total white blood cell (leukocyte)
counts, red blood cell counts, platelet counts and hemoglobin levels, were measured using an automated hematol-
ogy analyzer (Sysmex XT- 2000-I, Sysmex America, IL, USA). All blood samples of the participants were collected
after an overnight 12-hour fast at the same blood drawing station.
Physical fitness assessment. The time for completing a 3000-meter run test, which is a strong indicator
of the velocity at lactate (anaerobic) threshold, is used to evaluate the superiority of aerobic fitness25. The partici-
pants did not carry any objects during the test. All runs began at 4:00 PM and were performed on a flat rectangu-
lar playground only if there was no heavy rain and the risk coefficient of heat stroke (i.e., the outdoor temperature
(°C) × the outdoor relative humidity (%) × 0.1) was lower than 40. In addition, the number of push-ups per-
formed in 2 minutes and the number of sit-ups performed in 2 minutes were separately investigated for the supe-
riority of anaerobic fitness. These two procedures were scored in a standard manner by computer infrared sensing
systems. As the exercise tests are linked to rank promotion and awards, the best performance is considered the
fitness of each participant. All testing courses were monitored by the observing officers and video recorded. This
study was reviewed and approved by the Institutional Review Board of Mennonite Christian Hospital (No. 16-05-
008) in Hualien, Taiwan, and written informed consent was obtained from all participants. All procedures were
performed in accordance with the relevant guidelines and regulations.
Statistical analysis. Systemic low-grade inflammation was graded by equally sized quartiles of total leuko-
cyte counts within the normal limits as follows: the lowest leukocyte counts (4,000–5,499/mm3, N = 768), the sec-
ond lowest leukocyte counts (5,500–6,499/mm3, N = 1,015), the second highest leukocyte counts (6,500–7,499/
mm3, N = 842) and the highest leukocyte counts (7,499–9,999 × 103/mm3, N = 828). For the baseline character-
istics of each group, continuous data were expressed as the mean ± standard deviation (SD) and compared by
two-tailed t test. When the normality test (Kolmogorov-Smirnov) indicated non-normality, the Wilcoxon signed
rank test was used. Categorical data were expressed as numbers (%) and compared by χ2 test or Fisher’s exact test.
The differences in each exercise performance among those with the lowest, second lowest, second highest and
highest leukocyte counts were estimated by using analysis of covariance (ANCOVA), and the results are presented
as the mean ± standard error (SE). Multiple stepwise linear regressions were used to determine the correlation of
the second lowest, second highest, and highest leukocyte counts with each exercise performance with the lowest
leukocyte counts as the reference. In addition, given that the large sample size of the study could provide sufficient
power for subgroup analysis, multiple linear regressions for total leukocyte count and each exercise performance
were performed for those aged ≥30 years and those aged <30 years.
In model 1, age and service specialty were used to adjust the model. In model 2, body mass index was also
used. In model 3, the covariates in model 2 and waist circumference were used to adjust the model. In model 4,
smoking, alcohol intake status, and physical activity were also used. These potential confounders were chosen for
the models according to prior published associations between low-grade inflammation and fitness26,27. A 2-tailed
value of p < 0.05 was considered significant. SAS statistical software (SAS System for Windows, version 9.4; SAS
Institute, Cary, NC, United States) was used for all statistical analyses.

WBC counts WBC counts WBC counts WBC counts

0th–25th % 25th–50th % 50th–75th % 75th–100th %
Characteristics (n = 768) (n = 1,015) (n = 842) (n = 828) p-value
Age (years) 29.6 ± 5.9 29.1 ± 5.9 29.2 ± 5.9 29.3 ± 5.8 0.34
Specialty, %
Army 409 [53.3] 532 [52.4] 409 [48.6] 397 [47.9] 0.11
Navy 115 [20.2] 217 [21.4] 173 [20.5] 194 [23.4]
Air force 204 [26.5] 266 [26.2] 260 [30.9] 237 [28.6]
Body mass index (kg/m2) 24.3 ± 2.9 24.6 ± 3.0 25.0 ± 3.1 25.4 ± 3.2 <0.01
Waist circumference (cm) 81.8 ± 7.6 82.8 ± 7.8 83.7 ± 7.8 85.1 ± 8.1 <0.01
Systolic blood pressure (mmHg) 116.0 ± 12.9 118.1 ± 12.9 118.8 ± 13.1 120.4 ± 13.3 <0.01
Diastolic blood pressure (mmHg) 68.8 ± 9.9 70.5 ± 9.9 70.8 ± 10.2 71.9 ± 10.3 <0.01
Blood test
Total cholesterol (mg/dL) 171.4 ± 32.2 171.2 ± 33.5 176.6 ± 35.0 178.0 ± 34.3 <0.01
Serum triglyceride (mg/dL) 91.6 ± 66.8 107.3 ± 113.5 123.8 ± 104.8 131.2 ± 3.2 <0.01
Fasting glucose (mg/dL) 92.7 ± 9.8 93.7 ± 14.1 93.7 ± 12.1 94.3 ± 16.1 0.10
HDL-C (mg/dL) 49.8 ± 10.3 48.2 ± 9.4 47.3 ± 9.6 46.5 ± 9.4 <0.01
LDL-C (mg/dL) 103.1 ± 28.0 103.6 ± 29.3 107.7 ± 30.1 109.5 ± 30.4 <0.01
WBC counts (103/mm3) 4.9 ± 0.4 6.0 ± 0.3 7.0 ± 0.3 8.4 ± 0.7 <0.01
(WBC Range: Min-Max) 4.00–5.49 5.50–6.49 6.50–7.49 7.50–9.99
Hemoglobin, (g/dL) 15.0 ± 0.9 15.1 ± 1.0 15.2 ± 1.0 15.4 ± 1.0 <0.01
Alcohol intake status, %
Former or never 466 [60.7] 596 [58.7] 454 [53.9] 424 [51.2] <0.01
Current 302 [39.3] 419 [41.3] 388 [46.1] 404 [48.8]
Tobacco smoking status, %
Never smoker 535 [70.8] 692 [69.1] 485 [58.4] 418 [51.4] <0.01
Current smoker 221 [29.2] 310 [30.9] 346 [41.6] 395 [48.6]
Physical activity, %
Never or occasionally 151 [19.7] 204 [20.1] 192 [22.8] 174 [21.0] 0.75
1–2 times/week 287 [37.4] 387 [38.1] 314 [37.3] 307 [37.1]
≥ 3 times/week 330 [43.0] 424 [41.8] 336 [39.9] 347 [41.9]
Table 1. Baseline Characteristics of Study Participants Stratified by Leukocyte Counts (n = 3,453). Continuous
variables are expressed as mean ± SD (standard deviation), and categorical variables as n [%]. Abbreviations:
HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol, WBC: white blood cell.
Results
Baseline group characteristics. The baseline characteristics of each group are shown in Table 1. Within
the normal limits of total leukocyte counts, males with higher total leukocyte counts were found to have greater
body mass indexes, waist circumferences and atherogenic lipid profiles, and they had an increased prevalence of
tobacco smoking and alcohol beverage intake. Notably, the mean ages and physical activity of the participants in
the four groups were similar.
Group means comparisons. Table 2 reveals the ANCOVA results for testing the differences in exercise
performance among groups. There were differences in the numbers of push-ups and sit-ups in 2 minutes and the
time for a 3000-meter run test among the four groups in model 1 to model 3 (all overall p-values ≤0.01), indicat-
ing a trend towards low physical fitness and high leukocyte counts within normal limits hat is independent of age
and body adiposity. Compared with the lowest normal leukocyte counts, the second highest and highest normal
leukocyte counts had significantly lower numbers of push-ups in 2 minutes and longer times for a 3000-meter run
in model 4. There were no differences among the four groups in the number of sit-ups in 2 minutes in model 4.
Multiple linear regressions. The results of multiple linear regressions for each exercise performance, with
the second lowest, the second highest and the highest normal leukocyte counts in reference to the lowest normal
leukocyte counts, are shown in Table 3. The relationships of total leukocyte count and the performance for each
exercise in all models are in line with the findings presented in Table 2. In model 4, compared to the lowest nor-
mal leukocyte counts, the second highest and highest normal leukocyte counts were inversely correlated with a
longer time for a 3000-meter run test (β = 4.93 and 4.65, respectively) and fewer numbers of push-ups in 2 min-
utes (β = −0.59 and −0.56, respectively). However, there was no relationship between total leukocyte counts and
number of sit-ups in 2 minutes in model 4.
Subgroup analysis based on age. Table 4 reveals the results of multiple linear regressions for total leu-
kocyte counts and exercise performance in males aged ≥30 years and in males aged <30 years for each exercise.
The results were similar in the inverse association between the highest normal leukocyte counts and exercise

WBC counts 0th–25th WBC counts 25th–50th WBC counts 50th–75th WBC counts 75th–100th
p- p- p-
n Mean (SE) value n Mean (SE) value n Mean (SE) value n Mean (SE) p-value
2-min push-ups (numbers)
Model 1 765 50.4 (0.41) <0.01a 1009 50.2 (0.36) 0.49b 835 48.5 (0.40) <0.01c 818 47.4 (0.40) <0.01d
Model 2 764 50.4 (0.41) <0.01 a
1009 50.2 (0.36) 0.79b
833 48.5 (0.39) <0.01 c
814 47.4 (0.40) <0.01d
Model 3 763 50.4 (0.41) <0.01a 1002 50.1 (0.35) 0.89b 826 48.4 (0.39) 0.01c 808 47.4 (0.40) <0.01d
Model 4 752 50.4 (0.41) <0.01a 990 50.1 (0.35) 0.95b 817 48.5 (0.39) 0.03c 797 47.4 (0.40) <0.01d
2-min sit-ups (numbers)
Model 1 765 47.9 (0.28) <0.01a 1009 48.2 (0.25) 0.88b 839 47.2 (0.27) 0.03c 824 46.9 (0.27) <0.01d
Model 2 764 47.9 (0.28) <0.01a 1009 48.2 (0.25) 0.80b 837 47.2 (0.27) 0.05c 821 46.9 (0.27) <0.01d
Model 3 763 47.9 (0.28) 0.01 a
1002 48.1 (0.25) 0.73b
829 47.2 (0.27) 0.08c
815 46.9 (0.27) 0.01d
Model 4 752 47.9 (0.28) 0.11 a
990 48.2 (0.25) 0.56b
820 47.2 (0.27) 0.31c
804 46.9 (0.27) 0.11d
3000-m running (seconds)
Model 1 708 849.4 (2.55) <0.01a 934 852.6 (2.22) 0.09b 743 862.5 (2.49) <0.01c 723 872.6 (2.53) <0.01d
Model 2 707 849.5 (2.50) <0.01 a
934 852.6 (2.17) 0.28b
741 862.6 (2.44) <0.01 c
721 872.8 (2.78) <0.01d
Model 3 706 849.6 (2.50) <0.01 a
927 852.8 (2.17) 0.31b
734 862.5 (2.44) <0.01 c
716 872.8 (2.48) <0.01d
Model 4 697 849.7 (2.48) <0.01a 916 852.8 (2.15) 0.48b 725 862.3 (2.41) <0.01c 705 872.5 (2.47) <0.01d
Table 2. Differences in Each Exercise Performance between Different Leukocyte Count Groups. aOverall
p-value; bWBC: 0th-25th % vs. 25th-50th %; cWBC: 0th-25th % vs. 50th-75th %; dWBC: 0th-25th % vs. 75th-100th
%. Mean ± standard error (SE) for each exercise performance estimated using analysis of covariance with
adjustments for Model 1: age and service specialty; Model 2: the covariates in Model 1 and body mass index;
Model 3: the covariates in Model 2 and waist circumference; Model 4: the covariates in Model 3, alcohol intake
status, cigarette smoking status and weekly physical activity.
WBC Counts 25th–50th % WBC Counts 50th–75th % WBC Counts 75th–100th %

β 95% CI p-value β 95% CI p-value β 95% CI p-value
Model 1 −0.43 −1.49–0.62 0.42 −1.01 −1.57–−0.44 <0.01 −1.03 −1.41–−0.63 <0.01
Model 2 −0.23 −1.28–0.82 0.67 −0.81 −1.37–−0.24 <0.01 −0.77 −1.56–−0.38 <0.01
Model 3 −0.09 −1.23–0.94 0.86 −0.73 −1.28–−0.17 0.01 −0.65 −1.04–−0.26 <0.01
Model 4 −0.03 −1.05–0.99 0.95 −0.59 −1.15–−0.03 0.03 −0.56 −0.96–−0.17 <0.01
Model 1 0.06 −0.73–0.85 0.88 −0.44 −0.84–−0.04 0.03 −0.39 −0.65–−0.13 <0.01
Model 2 0.10 −0.69–0.89 0.80 −0.41 −0.81–−0.01 0.04 −0.36 −0.62–−0.95 <0.01
Model 3 0.17 −0.62–0.96 0.67 −0.36 −0.77–0.04 0.07 −0.31 −0.58–−0.05 0.02
Model 4 0.23 −0.55–1.01 0.56 −0.20 −0.62–0.20 0.31 −0.21 −0.48–0.05 0.11
3000-m run (seconds)
Model 1 5.46 −1.08–12.01 0.10 7.39 3.98–10.78 <0.01 8.10 5.62–10.59 <0.01
Model 2 3.45 −2.97–9.87 0.29 5.94 2.56–9.32 <0.01 6.03 3.58–8.49 <0.01
Model 3 3.19 −3.21–9.58 0.32 5.72 2.36–9.08 <0.01 5.79 3.33–8.24 <0.01
Model 4 2.58 −3.67–8.84 0.41 4.93 1.61–8.25 <0.01 4.65 2.20–7.10 <0.01
Table 3. Multiple Liner Regressions of Leukocyte Counts With Each Exercise Performance. Data are presented
as β-value and 95% confidence intervals (CI) using Pearson’s correlation coefficient for Model 1: age and service
specialty; Model 2: the covariates in Model 1 and body mass index; Model 3: the covariates in Model 2 and waist
circumference; Model 4: the covariates in Model 3, alcohol intake status, cigarette smoking status and weekly
physical activity.
performance for those aged ≥30 years and those aged <30 years in model 4. However, the inverse relationship
of the second highest normal leukocyte counts with exercise performance was not significant in those aged <30
years in model 4 for any exercise.
Discussion
Our principal findings were that systemic low-grade inflammation as reflected by total leukocyte counts within
normal limits was inversely associated with aerobic and anaerobic fitness in healthy young male military mem-
bers in Taiwan. Higher total leukocyte counts were correlated with longer times to complete a 3000-meter run, as
an evaluation of aerobic fitness, and fewer push-ups in 2 minutes, as an evaluation of anaerobic fitness.
Notably, both maximal aerobic and anaerobic fitness were decreased in male military members with
higher normal leukocyte counts (6,500–9,999/mm3) compared to those with lower normal leukocyte counts

WBC Counts 25th–50th % WBC Counts 50th–75th % WBC Counts 75th–100th %

N β-value 95% CI p-value β-value 95% CI p-value β-value 95% CI p-value
30–50 y/o 1787 −0.43 −1.66–0.80 0.49 −0.94 −1.58–−0.30 <0.01 −0.51 −0.94–−0.07 0.02
18–29 y/o 1640 0.59 −1.07–2.24 0.48 −0.06 −0.97–0.86 0.90 −0.58 −1.25–0.09 0.09
30–50 y/o 1794 0.08 −0.96–1.10 0.88 −0.50 −1.02–0.02 0.06 −0.24 −0.59–0.11 0.18
18–29 y/o 1643 0.50 −0.67–1.68 0.40 0.16 −0.45–0.76 0.61 −0.19 −0.59–2.14 0.35
3000-m run (seconds)
30–50 y/o 1598 4.42 −3.75–12.58 0.28 5.40 1.29–9.51 0.01 4.93 1.75–8.09 <0.01
18–29 y/o 1510 −0.36 −9.97–9.26 0.94 4.10 −1.19–9.40 0.12 4.14 0.32–7.95 <0.001
Table 4. Multiple Liner Regressions of Leukocyte Counts With Each Exercise Performance Stratified by Age.
Data are presented as β-value and 95% confidence intervals (CI) using Pearson’s correlation coefficient for
Model 4: age, service specialty, body mass index, waist circumference, alcohol intake status, cigarette smoking
status and weekly physical activity.
(4,000–6,499/mm3), indicating that a total leukocyte count of 6,500/mm3 could be thought of as the level at
which low-grade inflammation impairs both muscular strength and cardiorespiratory fitness. Although the types,
amount and frequency of daily physical activity were similar among military members, an inverse relationship
between total leukocyte counts within normal limits and the performance of 3000-m run and 2-minute push-up
tests remained remarkable. In addition, the inverse relationship was present independent of body adiposity, habit-
ual tobacco smoking and alcohol intake, which are the most common causes of low-grade inflammation and
impaired physical fitness among young adults free of chronic diseases7,8.
Numerous studies have revealed an association of low-grade inflammation with low physical activity, greater
body fat, insulin resistance and a higher risk of type 2 diabetes and cardiovascular disease28–32. These might be the
mechanisms for the inverse relationship between total leukocyte counts and cardiorespiratory fitness in middle-
and old-aged individuals in previous population studies13–16. However, most of the studies did not control for
baseline confounders, such as physical activity and habitual toxic agent use (e.g., smoking and alcohol) status.
Although some reports also revealed that levels of physical fitness were associated with C-reactive protein and C3
complement concentrations among young adults or prepubertal children28,33, most of the studies were limited by
a small sample size and by the method of model adjustment only controlling for body adiposity and not for other
confounding factors. The findings of this study were consistent with previous study results that total leukocyte
counts, which are highly related to C-reactive protein and C3 complement concentrations, could also be used for
evaluating the superiority of physical fitness in young adults.
With regard to the relationship between low-grade inflammation status and anaerobic fitness (muscular
strength), the inverse association was present in both the 2-minute push-up and 2-minute sit-up tests and was inde-
pendent of the demographic and anthropometric indexes. However, the relationship became nonsignificant for the
2-minute sit-up test with additional adjustment for physical activity and toxic agent use. The association differed for
the two anerobic exercise tests, possibly due to a variation in intensity of training and the amount of current tobacco
smoking and alcohol intake. In addition, we found that the threshold for a significant inverse association between
total leukocyte count and physical fitness was increased to 7.5 × 103/mm3 in the highest category in individuals aged
18–29 years. This finding could be the reason that a mild elevation in total leukocyte count at baseline was present in
the younger participants; the elevation might be caused by a greater immune response to environmental stimuli and
physical training rather than low-grade inflammation in the body, and thus, the association of the second highest
total leucocyte count with physical fitness was null in those males aged less than 30 years in the present study.
Our study had several strengths. First, both routine blood examinations and exercise tests were performed
strictly, and all of the procedures were performed in a standard manner. Second, since there were numerous
male military members included in the study, the power was sufficient to detect differences among groups. Third,
many unmeasured confounders in this study would be minimized as daily schedules in the military are similar.
In contrast, our study had some limitations. First, the study included only young physically fit male subjects,
and it is difficult to apply the results to young female subjects, older individuals, and those with overall average
or below-average fitness. Second, since the participation rate of male military members in the CHIEF study was
66.5%, we could not exclude the possibility of selection bias. Third, although many covariates were controlled at
baseline, the presence of potential confounders such as social/mental stress, medication use, and musculoskeletal
conditions, which might result in a bias, could not be avoided. Fourth, we did not have information regarding
the subtypes of leucocytes, and the total leukocyte count might vary with different situations, such as changes
in weather, despite blood samples being drawn in the same place with good air conditioning in accordance with
the rules for laboratory examinations. Finally, the statistical analyses failed to correct for multiple comparisons.
In conclusion, our findings suggested that there was an inverse association of total leukocyte counts within
normal limits and aerobic fitness as well as parts of anaerobic fitness in young male military members in Taiwan
who underwent rigorous training regularly. In addition, the temporal association between total leucocyte count
and physical fitness needs confirmation in longitudinal studies.
Received: 27 December 2019; Accepted: 21 March 2020;

Published: xx xx xxxx

References
1. Minihane, A. M. et al. Low-grade inflammation, diet composition and health: current research evidence and its translation. Br. J.
Nutr. 114, 999–1012 (2015).
2. Calder, P. C. Biomarkers of immunity and inflammation for use in nutrition interventions: International Life Sciences Institute
European Branch work on selection criteria and interpretation. Endocr. Metab. Immune Disord. Drug Targets. 14, 236–244 (2014).
3. Calder, P. C. et al. Inflammatory disease processes and interactions with nutrition. Br. J. Nutr. 101(Suppl 1), S1–45 (2009).
4. Hotamisligil, G. S., Shargill, N. S. & Spiegelman, B. M. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-
linked insulin resistance. Science. 259, 87–91 (1993).
5. Ouchi, N., Parker, J. L., Lugus, J. J. & Walsh, K. Adipokines in inflammation and metabolic disease. Nat. Rev. Immunol. 11, 85–97 (2011).
6. de Ferranti, S. & Mozaffarian, D. The perfect storm: obesity, adipocyte dysfunction, and metabolic consequences. Clin. Chem. 54,
945–955 (2008).
7. Ohshita, K. et al. Elevated white blood cell count in subjects with impaired glucose tolerance. Diabetes Care. 27, 491–496 (2004).
8. Gaydos, J. et al. Alcohol abuse and smoking alter inflammatory mediator production by pulmonary and systemic immune cells. Am.
J. Physiol. Lung Cell Mol. Physiol. 310, L507–518 (2016).
9. Wagner, E. Y. et al. Evidence for chronic low-grade systemic inflammation in individuals with agoraphobia from a population-based
prospective study. PLoS One. 10, e0123757 (2015).
10. Michopoulos, V., Powers, A., Gillespie, C. F., Ressler, K. J. & Jovanovic, T. Inflammation in Fear- and Anxiety-Based Disorders:
PTSD, GAD, and Beyond. Neuropsychopharmacology. 42, 254–270 (2017).
11. Jae, S. Y., Heffernan, K. S., Lee, M. K., Fernhall, B. & Park, W. H. Relation of cardiorespiratory fitness to inflammatory markers,
fibrinolytic factors, and lipoprotein(a) in patients with type 2 diabetes mellitus. Am. J. Cardiol. 102, 700–703 (2008).
12. Rana, J. S. et al. Increased level of cardiorespiratory fitness blunts the inflammatory response in metabolic syndrome. Int. J. Cardiol.
110, 224–230 (2006).
13. Kim, D. J. et al. A white blood cell count in the normal concentration range is independently related to cardiorespiratory fitness in
apparently healthy Korean men. Metabolism. 54, 1448–1452 (2005).
14. Ichihara, Y. et al. Higher C-reactive protein concentration and white blood cell count in subjects with more coronary risk factors
and/or lower physical fitness among apparently healthy Japanese. Circ. J. 66, 677–684 (2002).
15. Church, T. S. et al. Relative associations of fitness and fatness to fibrinogen, white blood cell count, uric acid and metabolic
syndrome. Int J. Obes Relat Metab Disord. 26, 805–813 (2002).
16. Carroll, S., Cooke, C. B. & Butterly, R. J. Plasma viscosity and its biochemical predictors: associations with lifestyle factors in healthy
middle-aged men. Blood Coagul. Fibrinolysis. 11, 609–616 (2000).
17. Lin, G. M. et al. Rationale and design of the cardiorespiratory fitness and hospitalization events in armed forces study in Eastern
Taiwan. World J. Cardiol. 8, 464–471 (2016).
18. Chen, Y. J. et al. Chronic hepatitis B, nonalcoholic steatohepatitis and physical fitness of military males: CHIEF study. World J.
Gastroenterol. 23, 4587–4594 (2017).
19. Tsai, K. Z. et al. Association of betel nut chewing with exercise performance in a military male cohort: the CHIEF study. J R Army
Med Corps. 164, 399–404 (2018).
20. Tsai, K. Z. et al. Association between mild anemia and physical fitness in a military male cohort: The CHIEF study. Sci. Rep. 9, 11165
(2019).
21. Chao, W. H., Su, F. Y., Lin, F., Yu, Y. S. & Lin, G. M. Association of electrocardio- graphic left and right ventricular hypertrophy with
physical fitness of military males: The CHIEF study. Eur J Sport Sci. 19, 1214–1220 (2019).
22. Chen, K. W. et al. Sex-Specific Association between Metabolic Abnormalities and Elevated Alanine Aminotransferase Levels in a
Military Cohort: The CHIEF Study. Int. J. Environ. Res. Public Health. 15, 545 (2018).
23. Lu, S. C. et al. Quantitative Physical Fitness Measures Inversely Associated With Myopia Severity in Military Males: The CHIEF
Study. Am. J. Mens Health. 13, 1557988319883766 (2019).
24. Su, F. Y., Wang, S. H., Lu, H. H. & Lin, G. M. Association of Tobacco Smoking with Physical Fitness of Military Males in Taiwan: The
CHIEF Study. Can. Respir. J. 2020, 5968189 (2020).
25. Grant, S., Craig, I., Wilson, J. & Aitchison, T. The relationship between 3 km running performance and selected physiological
variables. J Sports Sci. 15, 403–410 (1997).
26. Fernández, I., Canet, O. & Giné-Garriga, M. Assessment of physical activity levels, fitness and perceived barriers to physical activity
practice in adolescents: cross-sectional study. Eur. J. Pediatr. 176, 57–65 (2017).
27. Conway, T. L. & Cronan, T. A. Smoking, exercise, and physical fitness. Prev. Med. 21, 723–734 (1992).
28. Thomas, N. E. & Williams, D. R. Inflammatory factors, physical activity, and physical fitness in young people. Scand J Med Sci Sports.
18, 543–556 (2008).
29. Krekoukia, M. et al. Elevated total and central adiposity and low physical activity are associated with insulin resistance in children.
Metabolism. 56, 206–213 (2007).
30. Muscari, A. et al. Serum C3 is a stronger inflammatory marker of insulin resistance than C-reactive protein, leukocyte count, and
erythrocyte sedimentation rate: comparison study in an elderly population. Diabetes Care. 30, 2362–2368 (2007).
31. Vozarova, B. et al. High white blood cell count is associated with a worsening of insulin sensitivity and predicts the development of
type 2 diabetes. Diabetes. 51, 455–461 (2002).
32. Lin, G. M. et al. Low-Density Lipoprotein Cholesterol Concentrations and Association of High-Sensitivity C-Reactive Protein
Concentrations With Incident Coronary Heart Disease in the Multi-Ethnic Study of Atherosclerosis. Am. J. Epidemiol. 183, 46–52
(2016).
33. Ruiz, J. R., Ortega, F. B., Warnberg, J. & Sjöström, M. Associations of low-grade inflammation with physical activity, fitness and
fatness in prepubertal children; the European Youth Heart Study. Int J Obes (Lond). 31, 1545–1551 (2007).
Author contributions
Pei-Shou Chung wrote and drafted the paper; Kun-Zhe Tsai analyzed the data; Yen-Po Lin and Yu-Kai Lin made
critical revisions on the study; Gen-Min Lin conceived, designed and corresponded to the study.
Competing interests
The authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to G.-M.L.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
ative Commons license, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
© The Author(s) 2020

BMC Physiology BioMed Central
Research article Open Access

Delayed leukocytosis after hard strength and endurance exercise:
Aspects of regulatory mechanisms
Bjørn Audun Risøy1, Truls Raastad1, Jostein Hallén1, Knut T Lappegård3,
Kjersti Bæverfjord2, Astrid Kravdal2, Else Marie Siebke2 and
Haakon B Benestad*2
Address: 1The Norwegian University of Sport and Physical Education, Oslo, Norway, 2Institute of Basic Medical Sciences, Department of
Physiology, University of Oslo, Norway and 3Dept. of Medicine, Nordland Hospital, Bodø, Norway and University of Tromsø, Tromsø, Norway
Email: Bjørn Audun Risøy - bjornaudun@hotmail.com; Truls Raastad - Truls.Raastad@nih.no; Jostein Hallén - Jostein.Hallen@nih.no;
Knut T Lappegård - Knut.Lappegard@Nordlandssykehuset.no; Kjersti Bæverfjord - kjersti.baverfjord@norgespost.no;
Astrid Kravdal - astrid.kravdal@sensewave.com; Else Marie Siebke - e.m.siebke@basalmed.uio.no;
Haakon B Benestad* - h.b.benestad@basalmed.uio.no
* Corresponding author
Published: 11 December 2003 Received: 09 October 2003

Accepted: 11 December 2003
BMC Physiology 2003, 3:14
This article is available from: http://www.biomedcentral.com/1472-6793/3/14
© 2003 Risøy et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.
neutrophil granulocytesstress hormoneschemotaxiscytokineschemokinesCRPcomplement system
Abstract
Background: During infections, polymorphonuclear neutrophilic granulocytes (PMN) are mobilized from their bone
marrow stores, travel with blood to the affected tissue, and kill invading microbes there. The signal(s) from the
inflammatory site to the marrow are unknown, even though a number of humoral factors that can mobilize PMN, are
well known. We have employed a standardized, non-infectious human model to elucidate relevant PMN mobilizers. Well-
trained athletes performed a 60-min strenuous strength workout of leg muscles. Blood samples were drawn before,
during and just after exercise, and then repeatedly during the following day. Cortisol, GH, ACTH, complement factors,
high-sensitive CRP (muCRP), IL-6, G-CSF, IL-8 (CXCL8) and MIP-1β (CCL4) were measured in blood samples. PMN
chemotaxins in test plasma was assessed with a micropore membrane technique.
Results: About 5 hr after the workout, blood granulocytosis peaked to about 150% of baseline. Plasma levels of GH
increased significantly 30 min into and 5 min after the exercise, but no increase was recorded for the other hormones.
No significant correlation was found between concentrations of stress hormones and the subjects' later occurring PMN
increases above their individual baselines. Plasma G-CSF increased significantly – but within the normal range – 65 min
after the workout. IL-6 increased very slightly within the normal range, and the chemokines IL-8 and MIP-1β did not
increase consistently. However, we found a significant increase of hitherto non-identified PMN-chemotactic activity in
plasma 35, 50, and 60 min after the exercise. No systemic complement activation was detected, and (mu)CRP was within
the reference range at rest, 5 h and 23 h after the exercise. After endurance exercise, similar findings were made, except
for a cortisol response, especially from non-elite runners.
Conclusion: Apparently, a multitude of humoral factors can – directly or indirectly – mobilize PMN from marrow to
blood; some of the factors are, others are not known to be, chemotactic. Under different conditions, different selections
of these mobilizers may be used. In the late granulocytosis after heavy, long-lasting exercise a number of factors thought
capable of mimicking the granulocytosis of infectious diseases were apparently irrelevant.
Page 1 of 12
(page number not for citation purposes)
BMC Physiology 2003, 3 http://www.biomedcentral.com/1472-6793/3/14
Background the magnitude of the leukocytosis is apparently related to

Leukocytosis can result from increased cell traffic (mobili- the intensity and duration of exercise and seems to be
zation) from bone marrow to blood, demargination from most pronounced after strenuous endurance exercise [10].
the blood vessel walls (e.g. after intense physical exercise),
and decreased exit to tissues. We still do not have a thor- In the present study we have explored possible mecha-
ough understanding of the release of blood cells from the nisms behind the late occurring granulocytosis after an
bone marrow [1]. It has been suggested that similar or the exhaustive run (preliminary experiments) and after a sin-
same factors that control cell recruitment into inflamed gle bout of heavy strength exercise (main study), for the
tissues also regulate the mobilization from the bone mar- following reasons: (i) this granulocytosis may be a model
row [1]. However, exceptions have been found; for exam- of the granulocytosis of inflammatory diseases, (ii) hor-
ple, leukocyte mobilization depends on adhesive mones and other factors are known to mobilize PMN, but
interactions distinct from those formed during diapedesis their – or other, hitherto unknown or unsuspected factors'
of polymorphonuclear, neutrophilic granulocytes (PMN) – roles have not been fully characterized in a physiological
to inflamed tissues [2]. setting [3]. Animal experiments suggest that signals from
inflamed tissues are humoral and not nervous [11]. Some
PMN play a pivotal role in the defense against infections, of these humoral factors are adrenaline (epinephrine),
especially bacterial infections. A large reserve of bone mar- noradrenaline (norepinephrine), growth hormone (GH)
row PMN exists, which can be mobilized during infec- and cortisol [4,12], as well as plasma granulocyte colony-
tions or other types of inflammation. In the blood, about stimulating factor (G-CSF) and interleukin-6 (IL-6) [7].
half the PMN population is freely circulating and in We figured that comparisons between the time profiles of
dynamic equilibrium with so-called marginated PMN. blood concentrations of leukocytes on the one hand and
These latter PMN are found in the lungs, liver, spleen and suggested signal substances on the other might reveal
bone marrow – adherent to or at least in contact with the which signals be the important mobilizers in either or
endothelium of small blood vessels. PMN, as well as the both endurance and strength exercises. In particular, we
other types of leukocytes, can be rapidly released (demar- hypothesized that correlations between increased plasma
ginated) by for example adrenaline or physical exercise concentrations of putative regulators and the subsequent
[3]. PMN response to exercise might give important cues.
Numerous studies have established that intensive endur- Results

ance exercise (70–85% VO2max) induces a biphasic per- Endurance exercise study
turbation of the circulating leukocyte count [4]. Baseline values for all leukocytes and for sub-types neu-
Immediately post exercise, total leukocytes increase 50– trophils, lymphocytes and monocytes were all within the
100%, comprising all leukocyte types. Within 30 minutes normal range for both athletes and controls, but the neu-
of recovery, the lymphocyte count starts to decline to 30– trophil concentration was significantly higher for the ath-
60% below baseline levels, remaining low for 3 to 6 letes than for the controls (4.8 ± 0.8 vs. 3.6 ± 0.9 · 109/l).
hours. However, if the exercise has been moderate, e.g.
around 50% of VO2max, the lymphocyte count does not Both experimental groups had increased total leukocyte
decline in the recovery period [4,5]. In contrast, the con- counts after the 1.0–1.5 h run, statistically significant only
centration of PMN, after a transient decrease the first hour for the control group just after the exercise. There was also
after exercise, increases as cells are released from the bone a difference between the groups after 3 h (p < 0.01), with
marrow reserve, and stay high for several hours [6,7]. A the control group having the greater rise in leukocytes,
similar pattern of leukocyte concentration changes can be averages compared with baseline values being 211% and
seen after hard strength exercise [8]. 131%, respectively (Table 1). Rather than the early
leukocytosis, due to demargination of leukocytes, the aim
Most studies of the immune response to exercise have of this study was the late leukocytosis of exercise – as
concerned endurance exercise, while strength exercise has reflected by the 3-hour values – and its regulation.
received relatively little attention. In one of the few latter
reports, Nieman et al. [8] showed that a single bout of The 3-hour neutrophil blood concentration was signifi-
strength exercise, i.e. leg squat exercise to muscular failure, cantly raised (p < 0.05), the increase again being larger
can result in increased immune cell levels in blood, simi- among the controls than among the athletes (258% vs.
lar to those observed with intense endurance exercise, 142% of baselines; p < 0.01). Band neutrophils had then
despite only a moderate hormonal response. Further- increased to 700% of baseline (p < 0.01) for the controls
more, in a previous study, we have shown that strength and 250% of baseline (p = 0.055) for the athletes, with a
exercise performed by strength-trained athletes induced significant difference (p < 0.01) between the two groups
PMN accumulation in damaged muscles [9]. However, (Table 1). Monocyte concentration also increased to
Page 2 of 12
Table 1: Blood leukocyte subset responses to the 1–1.5 hour long run
Blood sampling Subjects Total leukocytes. Neutrophil Band neutrophils Lymphoc. Monocytes
Granuloc.
Before Athletes1 7.1 ± 1.1 4.8 ± 0.8 0.06 ± 0.05 1.3 ± 0.5 0.6 ± 0.2
Control2 6.0 ± 1.2 3.6 ± 0.9# 0.09 ± 0.05 1.2 ± 0.3 0.7 ± 0.3
Just after Athletes 8.5 ± 1.6 5.7 ± 1.0 0.09 ± 0.06 1.7 ± 0.8 0.8 ± 0.3
Control 9.9 ± 3.8* 7.1 ± 2.8* 0.21 ± 0.20 1.4 ± 0.7 0.8 ± 0.4
3 h after Athletes 9.2 ± 1.7* 6.7 ± 1.3* 0.15 ± 0.09 1.3 ± 0.4 0.8 ± 0.2
Control 12.2 ± 2.8* 8.7 ± 2.3* 0.63 ± 0.2*# 1.3 ± 0.2 1.2 ± 0.3*#
The values given are means ± SD in 109 cells/l. * = different from Pre, p < 0.05, # = difference between groups, p < 0.05; 1n = 7, 2n = 8.
Table 2: Changes in hormones and inflammatory mediators before, just after and 3 h after completion of the 1–1.5 hour long run
Blood sampling Subjects Cortisol (nmol.l-1) GH (mIU.l-1) IL-1β (pg. ml-1) G-CSF (pg.ml-1) CRP (mg.l-1)
Before Athletes1 290 ± 98 4.5 ± 10.3 6.5 ± 2.2 118 ± 39 0.7 ± 1.9
Control2 470 ± 221 1.5 ± 2.2 7.4 ± 1.4 103 ± 29 2.6 ± 2.7
Just after Athletes 395 ± 192 16.9 ± 7.0* 6.1 ± 1.6 120 ± 31 1.4 ± 3.8
Control 663 ± 132* 35.7 ± 14.0** 7.3 ± 2.0 119 ± 51 5.1 ± 3.6
3 h after Athletes 186 ± 153 3.1 ± 6.4 6.9 ± 2.2 125 ± 45 0.0 ± 0.0
Control 366 ± 258 2.5 ± 5.7 8.3 ± 3.0 100 ± 23 4.9 ± 2.2*
Values are means ± SD. * = p < 0.05; ** = p < 0.01, compared with baseline group value. 1n = 7 2n = 8
171% of baseline 3 h after the run in the control group (p the run, and in the last blood samples significantly lower
< 0.05), but not detectably for the athletes. No significant than in the controls (p < 0.01).
changes in the concentration of lymphocytes were meas-
ured shortly or 3 h after the run. The strength exercise study
At the first test workout (TW1) the subjects lifted (mean ±
Blood cortisol increased during the run to an average of SE) a total weight of 4495 ± 211 kg and at TW2 a total of
167% of baseline for the controls, 139% for the athletes, 4922 ± 270 kg (warm up excluded).
the rise being statistically significant (p < 0.05) only for
the former. Three hours after the run the cortisol concen- Baseline values of total blood leukocytes (5–6 · 10 9/l),
trations in both groups had fallen below baseline levels neutrophils (3–4 · 10 9/l), lymphocytes (~2 · 10 9/l) and
(70% of baseline for both groups taken together; Table 2). mixed cells (i.e. monocytes, eosinophils and basophils)
(~0.5 · 10 9/l) for both groups before TW1 and TW2 were
Both groups had a significant growth hormone (GH) all within normal ranges. There was no significant differ-
increase immediately after running (from 4.5 ± 10.3 ence between the groups. In general, the blood leukocyte
mIU.l-1 at baseline to 16.9 ± 7.0 mIU.l-1 for the athletes responses for both groups at TW1 and TW2 (after 2 weeks
and from 1.5 ± 2.2 mIU.l-1 to 37.5 ± 14.0 mIU.l-1 for the of heavy strength training for the heavy training (HT)
controls; Table 2), the levels declining to approximately group) were similar, but with a few exceptions: A signifi-
resting values 3 h later. Immediately after running, the cant reduction in lymphocytes 0–5 h and an increase in
control group had a significantly higher GH level than the mixed cells 5 h after exercise, as detailed below.
athletes (p = 0.02). IL-1β and G-CSF did not vary detecta-
bly, nor were there any significant differences between the Total leukocytes in both groups were increased 25–43%
groups (Table 2). At rest, CRP values were low (Table 2), compared with baseline 30 min into TW1 and TW2, and
indicating absence of inflammations and infections (ref- 18–20% 5 hr after the exercise bouts (p < 0.05). The con-
erence values: < 10 mg.l-1). The controls had an increase – centrations were not significantly different from pre-exer-
though within the reference range – both immediately cise levels 5–65 min and 23 h after the bouts. Moreover,
and 3 h after the exercise burst, only the latter being statis- there were no significant differences between TW1 and
tically significant (p < 0.05), however. The athletes' values TW2 concerning the total leukocyte time profiles.
were apparently unchanged from before to 0 and 3 h after
Page 3 of 12
Neutrophil concentrations in both groups (for TW1 and cortisol immediately after exercise and lymphocyte levels
TW2) were increased 25–26% compared with baseline 30 65 min later (TW1, r = -0.506; p = 0.054; TW2, r = - 0.147;
min into the exercise bouts. The concentrations in general p = 0.573) (Table 4). No correlations could be found asso-
rapidly approached baseline after the end of the exercise, ciating individual hormone levels to the later occurring
except for the HT group which showed a significant neu- neutrophilia (Table 4).
trophilia (p < 0.05) 5–65 min after TW2. Blood neu-
trophils then increased 48–63% above baseline in both Blood neutrophil migration increased when exposed to
groups, for TW1 and TW2 5 h after the exercise, before chemotactic substances in blood (i.e. 20 % plasma)
returning to pre-exercise levels at 23 h. No significant dif- drawn 35, 50 and 65 min after strength exercise, in com-
ferences in neutrophil counts between TW1 and TW2 were parison with pre-exercise plasma (p < 0.001; Fig. 2). The
observed, nor between the HT and normal training (NT) migration was not significantly different from pre-exercise
groups (Fig. 1). levels at 5 min, 20 min or 5 h after the bouts.
Lymphocytes in both groups (for TW1 and TW2), after the Positive correlations between individual changes in chem-
expected rise during exercise, fell 15–27% below pre-exer- otactic activity (peak neutrophil migration) and blood
cise levels 5–65 min after the bout (p < 0.01; Fig. 1). After neutrophil concentrations (% change from baseline) 5 h
two weeks of heavy training the HT group showed an after exercise were found after both TW1 (r = 0.630, p =
increased lymphocytopenia (p = 0.01 – 0.03) 0–5 h after 0.13, n = 7 subjects) and TW2 (r = 0.655, p = 0.056, n = 9
TW2, as compared with TW1. For TW2, blood lym- subjects). The failing statistical significance in these exper-
phocytes in the HT group were still below pre-exercise val- iments may have been due to the few subjects tested (type
ues at 23 h (p = 0.03; Fig. 1). II statistical error).
Mixed cell concentration for both groups also increased Plasma levels of IL-6 increased slightly – but within the
30 min into the workouts (p = 0.01 – 0.04; non-signifi- normal range – for all six subjects at 5 and 35 min after
cant for the NT group in TW1). The mixed cell concentra- exercise and for five of the six subjects at 50 and 65 min.
tion ran a variable course after exercise (Fig. 1). Five hours Measured as optical density of the ELISA assay, they
after TW1 the mixed cell levels had increased 37% above increased 25–30% above baseline levels, but the changes
baseline (p = 0.001) for the HT group, which was signifi- in absolute values were small and ranged from 0.72
cantly higher than after the heavy training period (TW2) to1.04 pg. ml-1. Plasma levels of G-CSF increased signifi-
(p = 0.008; Fig. 1). In further experiments, we observed a cantly from 11.4 ± 2.7 pg.ml-1 at baseline to 17.2 ± 4.6 pg.
47% increase in monocytes 5 h after a similar test workout ml-1 65 min after the workout (p < 0.05). The increases
for the NT group by using differential counting (p < 0.01; were again within the normal range, occurred in five of six
n = 6). subjects, and were not significantly different from base-
line at any other time point. In contrast to the endurance
Plasma levels of GH for both the NT and HT groups exercise study, G-CSF levels were here measured with high
increased significantly 30 min into and 5 min after TW1 sensitivity ELISA kits. We found no consistent increases in
and TW2 (Table 3). In contrast to the increased cortisol IL-8 or MIP-1β plasma concentrations. Positive, but statis-
levels observed after the endurance exercise, blood corti- tically non-significant, correlations were observed
sol in general tended to be reduced to below pre-exercise between blood neutrophil concentrations 5 h after
levels 30 min into and 5 min after the strength workouts exercise and the increase in G-CSF (r = 0.55, p > 0.26, n =
(Table 3). However, when the normal diurnal rhythm of 6) and IL-6 (r = 0.71, p = 0.11, n = 6).
reduced secretion of cortisol throughout the day was
taken into account, the reduction was probably not due to The CRP values were below the upper limit for the refer-
the exercise per se. Plasma levels of ACTH were apparently ence values at rest, 5 h and 23 h after a bout of heavy
unchanged 30 min into and 5 min after strength work- strength exercise (TW1), except for one subject's values
outs, compared with pre-exercise values (Table 3). How- (that were elevated at all time points, probably due to a
ever, after the heavy training we observed higher ACTH slight infection the days before the exercise).
levels during TW2 than TW1, 30 min after start of exercise
(p < 0.05; Table 3). Complement activation was assessed by measuring
C1inhibitor-C1rs complexes, C4bc, C3bBbP, C3bc and
A positive correlation was observed between individual TCC to detect activation of the classical, alternative, final
increases (% of baseline) in GH during exercise and neu- common and terminal pathways, respectively. We found
trophils 5 min after exercise (Table 4). An inconsistent no evidence of systemic complement activation being
and statistically non-significant negative correlation was involved in the mobilization of neutrophils from the
found between the individual changes (% of baseline) in bone marrow, hours after heavy strength exercise.
Page 4 of 12
NT group HT group
200 R1 200
180 R2 180
(% of baseline)
Neutrophils
160 160
140 140
120 120
100 100
80 80
60 60
B A 1 10 25 B A 1 10 25
180 180
R1
160 160
R2
(% of baseline)
Lymphocytes
140 140
120 120
100 100
80 80
60 60
B A 1 10 25 B A 1 10 25
180 R1 180
160 R2 160
(% of baseline)
Mixed cells
140 140
120 120
100 100
80 80
60 60
B A 1 10 25 B A 1 10 25
Time (hrs) Time (hrs)
Figure 1leukocytosis
Exercise
Exercise leukocytosis. Blood leukocyte subset responses to single bouts of strength exercise before (R1 = TW1) and after
(R2 = TW2) a 2-week period of high volume strength training for the HT (heavy training) and NT (normal training) groups.
Values are means ± SE. Note the logarithmic time axes; time B = before and A = 5 min after exercise.
Page 5 of 12
Table 3: Acute hormonal responses to single bouts of 6 RM strength exercise
Blood sampling Subjects Cortisol (nmol.l-1) GH (mIU.l-1) ACTH (ng.l-1)
HT group
Pre (08.00 h) TW1 513 ± 48 0.6 ± 0.2 45.7 ± 10.2
TW2 527 ± 34 0.6 ± 0.2 46.2 ± 10.5
NT group
TW1 441 ± 56 0.4 ± 0.1 42.6 ± 10.7
TW2 484 ± 39 0.5 ± 0.1 42.0 ± 7.0
HT group
Mid (09.30 h) TW1 428 ± 42 10.8 ± 5.1* 41.6 ± 6.2
TW2 492 ± 35 8.0 ± 3.8* 55.1 ± 7.4#
NT group
TW1 383 ± 45 1.5 ± 0.6 50.7 ± 12.0
TW2 381 ± 26* 11.5 ± 3.5*# 50.6 ± 15.1
HT group
5 m post (10.05 h) TW1 420 ± 32* 7.4 ± 1.8* 36.8 ± 4.5
TW2 473 ± 47 7.4 ± 2.9* 35.8 ± 4.3
NT group
TW1 410 ± 45 9.3 ± 3.2* 42.3 ± 7.5
TW2 318 ± 30* 12.0 ± 1.3* 38.3 ± 7.4
TW1 (before) and TW2 (after) a 2-week period of high volume strength training for the HT group and normal strength training (NT group). * =
different from baseline, p < 0.05, # = different from TW1, p < 0.05. Values are means ± SE.
Table 4: Correlations between individual serum levels of stress hormones and leukocyte deviations (% changes from baseline)
Hormone Leukocyte Time after exerc.(h) (hormones/leukoc.) Correlation coeff. (r) P value
Cortisol Neutrophils 0/0 – TW1 -0.49 0.06

0/0 – TW2 -0.32 0.21
Lymphocytes 0/1 – TW1 -0.51 0.05
Mixed cells 0/5 – TW1 -0.31 0.26
0/5 – TW2 0.31 0.25
GH Neutrophils During/0 – TW1 0.56 0.025
During/0 – TW2 0.76 0.001
The table shows correlations for the HT and NT groups taken together at various times after TW1 and TW2. All other correlations (e.g. cortisol
at 5 min vs. lymphocytes 65 min after, r = -0.15; p = 0.57) were <0.3 or >-0.3.
Discussion In the present study we failed to find a statistically signif-

In our endurance experiments both athletes and non- icant relationship between the delayed-onset neutrophilia
trained controls showed the well-known, late occurring and any of the possible mediators measured. However, we
leukocytosis, due to granulocytosis with release of band discovered a significant increase of (hitherto non-identi-
neutrophils, but the response was larger among the fied) granulocyte chemotaxins in plasma 30–60 min after
untrained runners. The latter also had a slight monocyto- the strength exercise bout. The individual increases in this
sis 3 h after the test run. The same pattern was seen after chemotactic activity (assessed as peak neutrophil migra-
the strength workout, where both blood granulocytes and tion) correlated positively with the late neutrophilia 5 h
monocytes at 5 hr had increased to about 150% of base- after both strength workouts. The borderline statistical sig-
line concentration, before returning to pre-exercise levels nificance in these experiments may have been due to the
at 23 h. The increase in the percentage of immature band number of subjects tested (type II statistical error); more
(nonsegmented) neutrophils (a shift to the left), in con- experiments have to be done. Moreover, we shall try to
nection with the delayed onset neutrophilia, provides identify the chemotaxin(s), since none of the mediators
evidence for the release of neutrophils from the bone discussed below are prime suspects as relevant chemotac-
marrow. tic factors in our assay.
Page 6 of 12
correlations between blood neutrophil concentration 5 h

60
* after exercise and the slight increase in G-CSF and IL-6 are
Neutrophil migration
50 * in line with these results.

(% change)
40 Others have shown that strenuous endurance exercise

30 * provokes increased levels of several cytokines in the
blood, in particular IL-1β, IL-6, IL-8, IL-10, TNF-α and
20 MIP-1β [20]. Also, intensive eccentric strength exercise led
10 to increased circulating levels of several cytokines (e.g. IL-
6, IL-10 and M-CSF), but the response seemed smaller and
0 occurred later after the workout, compared with strenuous
-1 5 20 35 50 65 300 endurance exercise [21]. The cytokine response therefore
Time (min) seems to be dependent on both duration and nature of the
activity [22].
plasma 2 migration stimulated by pre- and post-exercise
Neutrophil
Figure
Neutrophil migration stimulated by pre- and post- Cytokines are produced by a wide variety of cell types,
exercise plasma. The responses are shown as percentage exposed to a multitude of stimuli. The sources and mech-
change from chemotaxis obtained with pre-exercise plasma anisms of systemic cytokine release after exercise are at
in two strength exercise experiments, TW1 (open squares, n present largely unknown. A possible exception is the IL-6
= 9) and TW2 (filled diamonds, n = 10). Six migration cham- production by exercised and thereby slightly damaged
bers were used to assay chemotaxis in each batch of plasma. muscle [20]. Adrenaline infusion also raises blood IL-6
Values are means ± SE. * P < 0.05. levels [23]. Non-specific injury, as occurring during major
abdominal surgery, also raises blood levels of cytokines
like IL-6 and G-CSF [24].
Blood levels of cortisol and GH generally increase in

We found that plasma G-CSF increased significantly – but response to both prolonged, intensive endurance exercise
within the normal range – 65 min after the workout. IL-6 and heavy strength exercise – and more after endurance
increased very slightly within the normal range, whereas than strength exercise [8]. Cortisol has been shown to
the chemokines IL-8 (CXCL8) and MIP-1β (CCL4) did mobilize neutrophils from the bone marrow to the circu-
not increase consistently – nor did IL-1β after the endur- lation in some studies [3], while others have indicated
ance exercise. Possibly, synergism between G-CSF and IL- that corticosteroid-induced neutrophilia occurs primarily
6 (and enhanced (nor)adrenaline levels during the exer- by demargination of cells from the blood vessel walls,
cise) may explain at least part of the PMN mobilization with a minor contribution from the bone marrow [25].
and monocytosis. Infusions of G-CSF [13], IL-6 [14] and Injected growth hormone, resulting in blood concentra-
epinephrine [15] have all been reported to increase blood tions similar to those recorded during and after exercise,
levels of granulocytes and monocytes. Furthermore, IL-6 induced granulocytosis, but without affecting the blood
can prime hematopoietic [16] and hemic cells [17] for a mononuclear cells [26].
strengthened response to other cytokines. Our findings
are also in accordance with other studies, showing that IL- We found that plasma levels of GH were elevated midway
6 treatment mobilizes neutrophils from the marginated during and 5 min after the strength exercise, while no
pool into the circulation after 2–6 h [18]. In addition, ani- increases were recorded for the other stress hormones,
mal experiments have shown that both G-CSF [19] and IL- cortisol and ACTH. The GH increase during exercise corre-
6 [18] plays a role in the marrow release of neutrophil lated significantly with the blood neutrophil concentra-
lineage cells and in shortening the neutrophil transit time tion 5 min after exercise, but no other correlations were
through the bone marrow. found between each individual's hormone concentrations
and their neutrophil increases above baseline value.
In a recent study, Yamada et al. [7] found a significant
increase in plasma G-CSF immediately after a maximal Conflicting results have been reported concerning corti-
exercise on a treadmill, which correlated positively, not sol's role for the late neutrophilia. Some authors claimed
only with the neutrophil counts, but also with the stab cell that there is no positive correlation between blood con-
(band-nucleated granulocyte) counts 1 h post exercise. In centration of cortisol and the degree of second-phase
addition, the increased levels of plasma IL-6 1 h post exer- granulocytosis of exercise [27,28], while others did find a
cise correlated positively with the neutrophil counts 2 h positive correlation [29]. Our results from the strength
post exercise. Our respective positive, but non-significant, exercise are in line with the former view; our results from
Page 7 of 12
the endurance run with the latter. In conclusion, neither growth hormone that were larger among the non-athletes
cortisol alone, nor growth hormone, could explain both than among the orienteerers). Again, this is in contrast to
the delayed onset neutrophilia and the monocytosis, the findings by others [38,40]. We suggest that differences
found after our two kinds of exercise. concerning mode of exercise, in particular with respect to
its duration and intensity, together with the timing of
It has previously been demonstrated in animal experi- blood sampling, can explain at least some of the discrep-
ments that intravenously administered complement pro- ancies mentioned above.
teins (e.g. C5a, C3) can mobilize PMN from bone marrow
to blood [2,30,31]. Furthermore, a significant elevation of Conclusions
C5a has been reported after a marathon race, presumably We found no convincing relationship between the late
due to tissue damage activating the complement system mobilization of neutrophils and any of the suggested sig-
[32]. High-intensity cycling also allegedly activated the nal substances measured, even though "the jury is still
complement system [33]. It should be noted, however, out" concerning G-CSF and IL-6. Different mechanisms,
that care has to be taken to avoid activation during prepa- or different selections of mobilizers, may be involved in
ration of the plasma sample for analysis. On the other the delayed-onset neutrophilia in response to strength
hand, other studies found no changes or only small exercise, endurance exercise, and infections, since the
changes in blood levels of complement proteins after aer- (patho)physiological stress mechanisms differ considera-
obic exercise in trained and untrained persons [34,35]. bly between these inflammation models. The most
We found no evidence for increased complement activa- important finding of the present work may be (i) that an
tion after the bout of strength exercise, measuring activa- impressive granulocytosis may be provoked without
tion products at several points in the cascade reactions. detectable presence in blood of factors generally thought
However, this cannot totally rule out the possibility that to be mobilizers of the bone marrow PMN store and (ii)
the exercise induces local complement activation in the detection of chemotactic activity of unexplained ori-
exerted muscle, contributing to the local inflammatory gin in the participants' plasma 30–60 minutes after
response. exercise.
CRP has been claimed capable of increasing production of Methods

inflammatory cytokines from monocytes [36]. We found Subjects
a rapid blood increase in CRP, in the runners. This was Endurance exercise protocol
surprising, has to our knowledge not been reported ear- Seven athletes (3 females and 4 males, age (mean ± SD)
lier, and was probably caused by release of preformed pro- 27 ± 4 yr) from the national orienteering team and eight
tein from the liver. The finding must be validated in controls (3 females and 5 males, age 23 ± 1 yr), represent-
further studies. In the strength exercise study the micro- ing the normal population, were selected for the prelimi-
CRP (µCRP) was within the reference range at rest, 5 h and nary study. The controls had not been engaged in hard
23 h after the strength workout. Long-lasting, strenuous endurance training more than twice a week.
exercise has led to CRP increases – 16 h after a marathon
run [32]. Strength exercise protocol
Seventeen male students participated in the definitive
The present study showed that a 2-week period of exces- study. All subjects had performed recreational strength
sive strength exercise did not alter the resting immune sys- training for at least two years. They were randomly
tem – as judged by blood leukocyte levels. In general, also divided into a heavy training group (HT, age 26.4 ± 4.3 yr,
the exercise-induced leukocytosis was similar, before and n = 10) and a normal training group (NT, age 25.4 ± 3.5
after the training period, except for small deviations, like yr, n = 7) after a period of 4 weeks when all of them had
the HT group showing a slightly increased performed normal strength training.
lymphocytopenia after the second test workout. This is in
accordance with other studies suggesting that regularly The experiment complied with the current national laws,
performed, normal resistance exercise does not alter either and the Regional Ethics Committee of Norway approved
the resting immune system or the exercise-induced leuko- the protocol. Written informed consent was secured from
cytosis [37]. Moreover, trained orienteerers had no detect- all the participants.
ably lower baseline concentrations of blood granulocytes
than untrained controls. This is in contrast to findings by Experimental designs
others [38,39]. Moreover, we found a more marked acute In the endurance exercise study, subjects ran for 1–1.5
leukocytosis, late granulocytosis and monocytosis in non- hours. They were told to run to exhaustion and not to take
athletes (controls) than in the well-trained subjects (pos- breaks. Blood samples were taken before, immediately
sibly explainable by increases in blood cortisol and after and 3 h after the exercise bout. For practical reasons,
Page 8 of 12
the test runs could not be arranged at the same time of the liminary study measured with "Nycocard" (Nycomed
day in both groups of participants, so the first blood sam- Pharma A/S, Oslo, Norway), where the amount of CRP
ple was drawn at about Hr 11.00 for the control group and was estimated from a colour scale. In the strength exercise
Hr 17.00 for the athletes. study the CRP was measured by means of a high sensitiv-
ity immunoturbidimetric assay (Tina-quant®) on the
As detailed previously [41], all subjects in the strength Roche® Hitachi 917 automated clinical chemistry
exercise study had their first test workout after the 4 weeks analyzer. Internal quality controls at 2.5 and 13.9 mg· l-1
of normal strength training and the second test workout had an analytical CV (day-to-day variation) of 2.5 and
after an additional 2-week period with heavy training of 3.0%, respectively (courtesy of Dr. Lars Mørkrid, Rikshos-
leg extensors for the HT group only (Fig. 1). The standard- pitalet, Oslo). The effect of haemoconcentration, taking
ised test workouts (TW) were performed on the 4th day place during the exercise, was adjusted for by correcting all
after both the normal (TW1) and the heavy training values according to baseline hematokrit (Hct) values [i.e.
period (TW2). reported blood cell count = (measured cell no. · resting
Hct)/measured Hct].
Test workout
The strength workout consisted of squats, front-squats, Total and differential WBC counts in the strength exercise
and knee extensions. The subjects performed 3 sets of 6 study were performed with a Sysmex K-1000 (TOA Medi-
repetitions with a load that could be lifted for a maximum cal Electronics Co., Ltd., Kobe, Japan). Coefficient of
of 6 repetitions (100% of 6 RM) in all three exercises variation (CV) for replicate determination was <4% for
(same relative intensity in TW1 and TW2). Warm-up was neutrophils and lymphocytes. Changes in plasma vol-
accomplished as detailed previously [41]. Rest between umes were calculated from changes in the plasma total
sets and exercises were 3 min, except for 8 min between protein (TP) concentration. TP in plasma was measured
squats and front squats in order to draw a blood sample. with a Kodak Ektachem DT60 analyser (Eastman Kodak
The workouts lasted 62 min. Meals were served at the Company, NY, USA), CV<5%.
same time of the day during both standardised strength
workout trials. The subjects ate the same type and amount Hormones
of food before and after each trial. Cortisol and growth hormone were analysed in serum
samples, while ACTH was analysed in EDTA plasma sam-
Blood sampling ples. All measurements were performed at Hormone Lab-
In the endurance exercise study, blood samples (one 9-ml oratory, Aker University Hospital, Oslo, by commercially
EDTA-tube and two 9-ml vacutainers for serum analyses) available kits, as described in detail by Raastad et al. [41].
were collected just before, immediately after and 3 h after The coefficients of intra-assay (CVintra) and inter-assay
the exercise bout. (CVinter) variation were 4% and 8% for cortisol, 10% and
13% for GH, and 7% and 8% for ACTH.
In the strength exercise study, blood samples were col-
lected 30 minutes before the heavy strength exercise, 25 Complement factors
min into the workout, and 5, 20, 35, 50, 65 min and 5 and Enzyme immunoassays (EIA) were used for analysing
23 h after the workout (Fig. 3). Blood was drawn from an complement factors in EDTA plasma. Activation of the
antecubital vein into one 5-ml heparin, one 9-ml serum classical complement pathway was quantified with EIAs
and two 3-ml EDTA vacutainers. Heparin and EDTA tubes detecting C1rs-C1inhibitor complexes (C1rs-C1inh) and
were set on ice and within 30 min centrifuged at 1000 g C4bc, the latter also being indicative of the lectin path-
for 10 min at 4°C. Some EDTA plasma samples for analy- way. Both methods have been described in detail
sis of cytokines and chemokines were centrifuged once elsewhere [42,43], and the antibodies used were a kind
more to get rid of platelet microvesicles, 11000 g for 5 gift from Professor C.E. Hack, Amsterdam, The Nether-
min. Serum tubes stood 30–45 min in room temperature lands. Activation of the alternative pathway was detected
before centrifugation. Plasma and serum were stored at - by quantifying the alternative convertase C3bBbP in an
20°C until analysis. EIA described previously [44]. Activation of the final com-
mon pathway was recorded with an EIA using the mono-
Analysis of haematological and immune variables clonal antibody bH6, specific for a neo-epitope exposed
White blood cell (WBC) counts in the endurance exercise in C3b, iC3b and C3c, as previously described [45]. Acti-
study were performed with a coulter counter. Differential vation of the terminal pathway was quantified with an EIA
counts of 200 WBC were done on a May-Grünwald/ using the monoclonal antibody aE11 specific for C9
Giemsa stained blood smear from each sample, so that incorporated in the terminal sC5b-9 complex (TCC), as
absolute numbers of the white blood cell subtypes could described previously [46].
be calculated. C-Reactive Protein (CRP) was in this pre-
Page 9 of 12
TW1 responses TW2 responses
Normal Heavy vs. normal training

training
0 5 6 7 8 9
Time (weeks)
Day 1: p = blood samples Day 2:

p p ppppp p p
TW1/TW2 Lunch Dinner Breakfast
08.00 10.00 12.00 14.00 16.00 08.00 10.00
Time of day (hours)
Figure 3and test session time lines of the strength exercise study
Training
Training and test session time lines of the strength exercise study. Experimental design (upper panel): The acute hae-
matological and immune responses to single bouts of 6 RM strength exercise was measured before (TW1) and after (TW2) a
2-week period of high volume strength training for the HT group and normal strength training for the NT group. Lower panel
illustrates timing of blood samples and meals in TW1 and TW2.
Cytokine and chemokine analysis Benestad [47]. Heparinized (20 IU.ml-1) blood was drawn
IL-1β and G-CSF in serum collected in the preliminary from healthy laboratory personnel and separated accord-
study were immuno-assayed with Quantikine™ ELISA- ing to a method originally described by Boyum [48],
kits, according to the instructions from the manufacturer slightly modified. Briefly, leukocytes were isolated after
(R&D System, Oxon, UK). Serum IL-6, G-CSF, IL-8 and erythrocyte aggregation and sedimentation with hydrox-
MIP-1β concentrations were in the strength exercise study yethyl starch (HES) (Fresenius AG, Bad Homburg, Ger-
immuno-assayed for six subjects at TW1. A high sensitivity many) and centrifugation of the leukocyte supernatant at
kit was used for analysing G-CSF. 400 g for 10 min. The leukocytes were washed once and
resuspended to a final cell concentration of 7 · 109/l in
Leukocyte chemotaxis medium. The migration assembly is an acrylic chamber
Leukocyte chemotactic migration was assessed with a with a 140-µm thick micropore membrane (Sartorius SM
micropore membrane technique described by Grimstad & 11324) with an average pore diameter of 5 µm as floor,
Page 10 of 12
immersed in a retrieval compartment. The cells were inoc- 2. Jagels MA, Chambers JD, Arfors KE, Hugli TE: C5a- and tumor
necrosis factor-alpha-induced leukocytosis occurs independ-
ulated into the acrylic chamber, and the leukocytes that ently of beta 2 integrins and L-selectin: differential effects on
had migrated to the retrieval compartment after 2 h, in the neutrophil adhesion molecule expression in vivo. Blood 1995,
presence of different chemoattractants or other modula- 85:2900-909.
3. Benestad HB, Laerum OD: The neutrophilic granulocyte. Curr
tors of migration, were brought into suspension by vigor- Top Pathol 1989, 79:7-36.
ous shaking and counted with a coulter counter. More 4. Pedersen BK, Rohde T, Ostrowski K: Recovery of the immune
than 97% of the migrated leukocytes during 2 h of incu- system after exercise. Acta Physiol Scand 1998, 162:325-32.
5. Pedersen BK: Influence of physical activity on the cellular
bation have been shown to be neutrophils [46]. In the immune system: Mechanisms of action. Int J Sports Med
present experiments we assayed chemotaxins present in 1991:23-29.
6. Suzuki K, Totsuka M, Nakaji S, Yamada M, Kudoh S, Liu Q, Sugawara
20% v/v test plasma. K, Yamaya K, Sato K: Endurance exercise causes interaction
among stress hormones, cytokines, neutrophil dynamics,
Statistics and muscle damage. J Appl Physiol 1999, 87:1360-67.
7. Yamada M, Suzuki K, Kudo S, Totsuka M, Nakaji S, Sugawara K:
In the pre-study, arithmetic mean values with their stand- Raised plasma G-CSF and IL-6 after exercise may play a role
ard deviations (SD) are used to characterize dispersion of in neutrophil mobilization into the circulation. J Appl Pysiol
2002, 92:1789-94.
the data. The Mann-Whitney test was applied to analyse 8. Nieman DC, Henson DA, Sampson CS, Herring JL, Suttles J, Conley
the difference between two groups of data (two-sided test; M, Stone HM, Butterworth DE, Davis JM: The acute immune
statistical significance level 0.05). In the strength exercise response to exhaustive resistance exercise. Int J Sports Med
1995, 16:322-328.
study paired t-tests and when indicated Wilcoxon Signed- 9. Raastad T, Risøy BA, Benestad HB, Fjeld JG, Hallén J: Temporal
Ranks test were used to identify exercise induced hormo- relationship between leukocyte accumulation in muscles and
nal, leukocyte, cytokine and chemotactic responses to the halted recovery 10–20 h after strength exercise. J Appl Physiol
2003, 285:2503-2509.
two test workouts. To compensate for inter-experimental 10. Natale VM, Brenner IK, Moldoveanu AI, Vasiliou P, Shek P, Shephard
variability, the non-parametric Wilcoxon-van Elteren [49] RJ: Effects of three different types of exercise on blood leuko-
cyte count during and following exercise. Sao Paulo Med J 2003,
test was used to calculate the significance of differences 121:9-14.
between test and control groups of several experiments 11. Benestad HB, Strom-Gundersen I, Iversen OP, Haug E, Nja A: No
taken together. This procedure allows us to divide the neuronal regulation of murine bone marrow function. Blood
1998, 91:1280-7.
sample into blocks and then combine the information 12. Pedersen BK, Toft AD: Effects of exercise on lymphocytes and
from each of the blocks to arrive at a probability for the cytokines. Br J Sports Med 2000, 34:246-51.
entire set of data. This test becomes identical to Wilcoxon 13. Ulich TR, del Castillo J, McNiece IK, Yin SM, Irwin B, Busser K, Guo
KZ: Acute and subacute hematologic effects of multi-colony
Signed-Ranks test with n = 1+1 in all groups. We also stimulating factor in combination with granulocyte colony-
examined selected bivariate relationships using a Pearson stimulating factor in vivo. Blood 1990, 75:48-53.
14. Asano S, Okano A, Ozawa K, Nakahata T, Ishibashi T, Koike K,
Product moment correlation coefficient. Tabulated data Kimura H, Tanioka Y, Shibuya A, Hirano T: In vivo effects of
are given as means ± SE. Statistical significance was set at recombinant human interleukin-6 in primates: stimulated
p ≤ 0.05. production of platelets. Blood 1990, 75:1602-5.
15. Richardson RP, Rhyne CD, Fong Y, Hesse DG, Tracey KJ, Marano
MA, Lowry SF, Antonacci AC, Calvano SE: Peripheral blood leuko-
Authors' contributions cyte kinetics following in vivo lipopolysaccharide (LPS)
administration to normal human subjects. Influence of elic-
BAR performed the chemotaxis experiments, part of the ited hormones and cytokines. Ann Surg 1989, 210:239-45.
strength exercise experiment, and wrote the manuscript; 16. Ikebuchi K, Wong GG, Clark SC, Ihle JN, Hirai Y, Ogawa M: Inter-
TR and JH performed the strength exercise experiments; leukin 6 enhancement of interleukin 3-dependent prolifera-
tion of multipotential hemopoietic progenitors. Proc Natl Acad
KTL supervised the complement analyses; KB and AK per- Sci USA 1987, 84:9035-9.
formed the endurance exercise experiments; EMS super- 17. Kharazmi A, Nielsen H, Rechnitzer C, Bendtzen K: Interleukin 6
vised the chemotaxis experiments and designed one of the primes human neutrophil and monocyte oxidative burst
response. Immunol Lett 1989, 21:177-84.
graphs; HBB conceived of parts of the study, and partici- 18. Suwa T, Hogg JC, English D, van Eeden SF: Interleukin-6 induces
pated in its design, coordination, analysis and writing. All demargination of intravascular neutrophils and shortens
their transit in marrow. Am J Physiol Heart Circ Physiol 2000,
authors read and approved the final manuscript. 279:2954-60.
19. Price TH, Chatta GS, Dale DC: Effect of recombinant granulo-
Acknowledgements cyte colony-stimulating factor on neutrophil kinetics in nor-
mal young and elderly humans. Blood 1996, 88:335-40.
Support to HBB from the Norwegian Cancer Society, the Research Council 20. Pedersen BK, Steensberg A, Fischer C, Keller C, Ostrowski K, Schjer-
of Norway, and Anders Jahre's Foundation for the Promotion of Science is ling P: Exercise and cytokines with particular focus an muscle-
gratefully acknowledged. We also thank Inger Strøm-Gundersen for excel- derived IL-6. Exerc Immunol Rev 2001, 7:18-31.
lent technical assistance and Lars Mørkrid for the high sensitivity CRP anal- 21. Smith LL, Anwar A, Fragen M, Rananto C, Johnson R, Holbert D:
Cytokines and cell adhesion molecules associated with high-
yses. Grethe Bergseth, Dorte Christensen and Hilde Fure assisted in intensity eccentric exercise. Eur J Appl Physiol 2000, 82:61-67.
performing the complement analyses. 22. Suzuki K, Nakaji S, Yamada M, Liu Q, Kurakake S, Okamura N, Kumae
T, Umeda T, Sugawara K: Impact of a competitive marathon
References race on systemic cytokine and neutrophil responses. Med Sci
Sports Exerc 2003, 35:348-55.
1. Opdenakker G, Fibbe WE, Van Damme J: The molecular basis of 23. Sondergaard SR, Ostrowski K, Ullum H, Pedersen BK: Changes in
leukocytosis. Immunol Today 1998, 19:182-89. plasma concentrations of interleukin-6 and interleukin-1
Page 11 of 12
receptor antagonists in response to adrenaline infusion in 45. Garred P, Mollnes TE, Lea T: Quantification in enzyme-linked
humans. Eur J Appl Physiol 2000, 83:95-8. immunosorbent assay of a C3 neoepitope expressed on acti-
24. Kato M, Suzuki H, Murakami M, Akama M, Matsukawa S, Hashimoto vated human complement factor C3. Scand J Immunol 1988,
Y: Elevated plasma levels of interleukin-6, interleukin-8, and 27:329-35.
granulocyte colony-stimulating factor during and after 46. Mollnes TE: Analysis of in vivo complement activation,. in :
major abdominal surgery. J Clin Anesth 1997, 9:293-8. Weir's Handbook of Experimental Immunology Edited by: Herzenberg LA,
25. Nakagawa M, Terashima T, D'yachkova Y, Bondy GP, Hogg JC, van Weir DM, Herzenberg L, Blackwell C. Boston, Blackwell Science;
Eeden SF: Glucocorticoid-induced granulocytosis – Contribu- 1997:78.1-78.8.
tion of marrow release and demargination of intravascular 47. Grimstad IA, Benestad HB: A new assay for leukocyte chemo-
granulocytes. Circulation 1998, 98:2307-13. taxis using cell retrieval, electronic particle counting and
26. Kappel M, Hansen MB, Diamant M, Jorgensen JO, Gyhrs A, Pedersen flow cytometry. J Immunol Methods 1982, 49:215-33.
BK: Effects of an acute bolus growth hormone infusion on the 48. Boyum A: Separation of leukocytes from blood and bone
human immune system. Horm Metab Res 1993, 25:579-85. marrow. Scand J Clin Lab Invest 1968, 21:77-89.
27. Hansen JB, Wilsgard L, Østerud B: Biphasic changes in leukocytes 49. Van Elteren P: On the combination of independent two sample
induced by strenuous exercise. Eur J Appl Physiol 1991, 62:157-61. tests of Wilcoxon. Bull Inst Int Stat 1960, 37:351.
28. Shinkai S, Watanabe S, Asai H, Shek PN: Cortisol response to
exercise and post-exercise suppression of blood lymphocyte
subset counts. Int J Sports Med 1996, 17:597-603.
29. McCarthy DA, Macdonald I, Grant M, Marbut M, Watling M, Nichol-
son S, Deeks JJ, Wade AJ, Perry JD: Studies of the immediate and
delayed leukocytosis elicted by brief (30-min) strenuous
exercise. Eur J Appl Physiol 1992, 64:513-517.
30. Kubo H, Graham L, Doyle NA, Quinlan WM, Hogg JC, Doerschuk
CM: Complement fragment-induced release of neutrophils
from bone marrow and sequestration within pulmonary cap-
illaries in rabbits. Blood 1998, 92:283-90.
31. Saito H, Lai J, Rogers R, Doerschuk CM: Mechanical properties of
rat bone marrow and circulating neutrophils and their
responses to inflammatory mediators. Blood 2002, 99:2207-13.
32. Castell LM, Poortmans JR, Leclercq R, Brasseur M, Duchateau J, New-
sholme EA: Some aspects of the acute phase response after a
marathon race, and the effects of glutamine
supplementation. Eur J Appl Physiol 1997, 75:47-53.
33. Camus G, Duchateau J, Deby-Dupont G, Pincemail J, Deby C,
Juchmes-Ferir A, Feron F, Lamy M: Anaphylatoxin C5a produc-
tion during short-term submaximal dynamic exercise in
man. Int J Sports Med 1994, 15:32-35.
34. Nieman DC, Tan SA, Lee JW, Berk LS: Complement and immu-
noglobulin levels in athletes and sedentary controls. Int J Sports
Med 1989, 10:124-28.
35. Wolach B, Eliakim A, Gavrieli R, Kodesh E, Yarom Y, Schlesinger M:
Aspects of leukocyte function and the complement system
following aerobic exercise in young female gymnasts. Scand J
Med Sci Sports 1998, 8:91-97.
36. Ballou SP, Lozanski G: Induction of inflammatory cytokine
release from cultured human monocytes by C-reactive
protein. Cytokine 1992, 4:361-8.
37. Simonson SR: The immune response to resistance exercise. J
Strength Cond Res 2001, 15:378-84.
38. Ferry A, Picard F, Duvallet A, Weill B, Rieu M: Changes in blood
leukocyte populations induced by acute maximal and
chronic submaximal exercise. Eur J Appl Physiol 1990, 59:435-42.
39. Nieman DC, Buckley KS, Henson DA, Warren BJ, Suttles J, Ahle JC,
Simandle S, Fagoaga OR, Nehlsen-Cannarella SL: Immune function
in marathon runners versus sedentary controls. Med Sci Sports
Exerc 1995, 27:986-92.
40. Ndon JA, Snyder AC, Foster C, Wehrenberg WB: Effects of
chronic intense exercise training on the leukocyte response
to acute exercise. Int J Sports Med 1992, 13:176-82.
41. Raastad T, Glomsheller T, Bjoro T, Hallen J: Recovery of skeletal
muscle contractility and hormonal responses to strength Publish with Bio Med Central and every
exercise after two weeks of high-volume strength training.
Scand J Med Sci Sports 2003, 13:159-68. scientist can read your work free of charge
42. Fure H, Nielsen EW, Hack CE, Mollnes TE: A neoepitope-based "BioMed Central will be the most significant development for
enzyme immunoassay for quantification of C1-inhibitor in disseminating the results of biomedical researc h in our lifetime."
complex with C1r and C1s. Scand J Immunol 1997, 46:553-7.
43. Wolbink GJ, Bollen J, Baars JW, ten Berge RJ, Swaak AJ, Paardekooper Sir Paul Nurse, Cancer Research UK
J, Hack CE: Application of a monoclonal antibody against a Your research papers will be:
neoepitope on activated C4 in an ELISA for the quantifica-
tion of complement activation via the classical pathway. J available free of charge to the entire biomedical community
Immunol Methods 1993, 163:67-76. peer reviewed and published immediately upon acceptance
44. Mollnes TE, Brekke O-L, Fung M, Bergseth G, Christiansen D, Fure H,
Videm V, Lappegård KT, Köhl J, Lambris JD: Essential role of the cited in PubMed and archived on PubMed Central
C5a receptor in E. Coli-induced oxidative burst revealed by a yours — you keep the copyright
novel lepirudin based human whole blood model of
inflammation. Blood 2002, 100:1869-1877. Submit your manuscript here: BioMedcentral
http://www.biomedcentral.com/info/publishing_adv.asp
Page 12 of 12

Accepted Manuscript: 10.1016/j.physbeh.2018.06.008

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Accepted Manuscript: 10.1016/j.physbeh.2018.06.008

Uploaded by

Copyright:

Available Formats

Accepted Manuscript

Lymphocytes and monocytes egress peripheral blood within

Bridgette V. Rooney, Austin B. Bigley, Emily C. LaVoy, Mitzi

Lymphocytes and monocytes egress peripheral blood within

Richard J. Simpson, PhD FACSM

Department of Nutritional Sciences

The University of Arizona

1177 E. Fourth Street

Room 308, Shantz Building

Tucson, Arizona 85721, USA

Key Words: Exercise Immunology, Leukocytosis, T-Cell, NK-Cell, Neutrophilia,

medications/supplements known to affect the immune system or cardiovascular system.

1.2.2 Experimental protocol:

(IgG1, TULY56), PE-conjugated anti-CD4 (IgG2b, OKT4), PerCy5-conjugated anti-CD8 (IgG1,

determined by multiplying the percentage of all gated cells (lymphocytes or monocytes)

1.2.4 Statistical Analysis

monocyte subtypes (Table 3).

influenced by exercise induced increases in both glucocorticoids [22,36,37,42] and a myriad of

Immun. 23 (2009) 823–9. doi:10.1016/j.bbi.2009.03.003.

J.A. Bosch, Acute exercise mobilises CD8+ T lymphocytes exhibiting an effector-memory

Pedersen, Splenectomy impairs lymphocytosis during maximal exercise., Am. J. Physiol.

[21] S. Dimitrov, T. Lange, J. Born, Selective mobilization of cytotoxic leukocytes by

induced cortisol on CCR2 expression on human monocytes, Brain. Behav. Immun. 22

after exercise, J. Appl. Physiol. 122 (2017) 1077 LP-1087.

[39] F. Zaldivar, J. Wang-Rodriguez, D. Nemet, C. Schwindt, P. Galassetti, P.J. Mills, L.D.

Pedersen, Interleukin-6 production in contracting human skeletal muscle is influenced by

response relationship between acute resistance-exercise and the immune system:

exercise: aspects of regulatory mechanisms., BMC Physiol. 3 (2003) 14.

Seixas, M.J. Laires, Sex-based effects on immune changes induced by a maximal

Physical Characteristics Mean SD Range

accordance with post-hoc Bonferroni correction, p<.05.

Recovery Time Point

Lymphocytes 0.599 0.454 0.187 0.124 0.434 -0.071

Granulocytes -0.028 0.133 0.195 0.432 0.208 0.137

Classical Monocytes -0.414 -0.555 0.276 -0.008 -0.134 -0.147

Intermediate Monocytes -0.469 -0.27 0.057 -0.076 0.082 -0.560

Pro-Inflammatory Monocytes -0.367 -0.336 0.232 0.105 0.498 -0.420

time point during exercise recovery, p<.05.

 Lymphocytes and monocytes egress blood immediately upon cessation of acute

OPEN Association between Leukocyte

Scientific Reports | (2020) 10:6082 | https://doi.org/10.1038/s41598-020-63147-9 1

Scientific Reports | (2020) 10:6082 | https://doi.org/10.1038/s41598-020-63147-9 2

WBC counts WBC counts WBC counts WBC counts

Scientific Reports | (2020) 10:6082 | https://doi.org/10.1038/s41598-020-63147-9 3

WBC Counts 25th–50th % WBC Counts 50th–75th % WBC Counts 75th–100th %

Scientific Reports | (2020) 10:6082 | https://doi.org/10.1038/s41598-020-63147-9 4

WBC Counts 25th–50th % WBC Counts 50th–75th % WBC Counts 75th–100th %

Received: 27 December 2019; Accepted: 21 March 2020;

Scientific Reports | (2020) 10:6082 | https://doi.org/10.1038/s41598-020-63147-9 5

Scientific Reports | (2020) 10:6082 | https://doi.org/10.1038/s41598-020-63147-9 6

© The Author(s) 2020

Scientific Reports | (2020) 10:6082 | https://doi.org/10.1038/s41598-020-63147-9 7

Research article Open Access

Published: 11 December 2003 Received: 09 October 2003

neutrophil granulocytesstress hormoneschemotaxiscytokineschemokinesCRPcomplement system

Background the magnitude of the leukocytosis is apparently related to

Numerous studies have established that intensive endur- Results

Table 3: Acute hormonal responses to single bouts of 6 RM strength exercise

Blood sampling Subjects Cortisol (nmol.l-1) GH (mIU.l-1) ACTH (ng.l-1)

Cortisol Neutrophils 0/0 – TW1 -0.49 0.06

Discussion In the present study we failed to find a statistically signif-

correlations between blood neutrophil concentration 5 h

50 * in line with these results.