Fourteen male, well-trained XC skiers volunteered to participate in this study (mean (SD); age: 25 (4) years, height: 183 (7) cm, and body mass: 77 (6) kg). The participants were recruited by personal communication with competing or former competing well-trained cross-country skiers. The inclusion criteria were 18–40 years old with \(\dot\)O2max > 60 ml·kg−1·min−1 and being experienced in classic diagonal treadmill roller-skiing. Exclusion criteria were splenectomy or any known disease or illness. Calculations of statistical power on the main variables were made a priori, after which decision was made to recruit a maximum of 15 participants to obtain a minimum of 12 participants. After responding to the first contact and agreeing to give out their email addresses, an information sheet for participants was distributed, and on arrival to the laboratory, an informed consent form was signed. This study was conducted in accordance with the Declaration of Helsinki and ethical approval was received through the regional review board in Umeå, Sweden (dnr 2020-00044).
Study designThe current study design included three parts divided over 2 days. The first day (test day 1) consisted of an apnea test and a \(\dot\)O2max test. The second day (test day 2), 2 to 10 days later, consisted of three submaximal intensity levels on a roller-skiing treadmill. The order of the intensity levels was randomized, and individual levels were calculated to correspond to 55, 70, and 85% of the participants’ measured \(\dot\)O2max.
Procedures and measurementsOn arrival at the laboratory, all participants were informed about the procedure and they signed the informed consent, after which measurements of body mass on a precision scale (Seca 764, Hamburg, Germany) and height were collected with a stadiometer attached to a wall. They were then equipped with a peripheral venous catheter, typically in the basilic or cephalic vein in the right arm fold. Seated down on a cushioned chair, they started the first resting period and filled out the health form, confirming that they felt completely healthy at the time. After 20 min of sitting rest, the first baseline splenic measurements and blood samples were collected.
Blood samplingThe venous blood samples were drawn with a 5-ml syringe, after which it was manually transferred into test tubes and cuvettes. A saline solution was used to flush the catheter and a waste sample of 1 ml was drawn from the arm before each sample. At each sampling occasion, a sample of 4 ml blood was drawn and transferred into three kinds of test tubes. First, a 500-µl sample was taken for analysis in a Horriba Micros 60 hematology analyzer (Diamond Diagnostic, Holliston, MA, US). Second, a blood lactate (Hla) sample (20 µl) was taken and measured with a Biosen S-line (EKF diagnostic GmbH, Magdeburg, Germany), which was calibrated with a standard solution of Hla (12 mmol·L−1) prior to each analysis. Third, the remainder of the sample (~ 3.5 ml) was transferred into a 3.5-ml vacutainer with a coagulant solution and analyzed for serum albumin (S-albumin) and total protein content (S-protein) with a Cobas Pro equipment (Roche Diagnostics, Indianapolis, IN, US). Serum S-protein and S-albumin concentration levels were analyzed to identify the hemoconcentration changes due to possible plasma volume changes.
Splenic measurementsSpleen volume was measured from the dorsal side via ultrasonic imaging (M-Turbo Ultrasound system, FUJIFILM SonoSite Inc., Bothell, WA) with the probe: C60x/5–2 MHz (SonoSite Inc., Bothell, WA, USA), by an experienced sonographer (PH). Two ultrasonic images were recorded each minute for determination of the following maximal three-axial diameters: length (L), width (W), and thickness (T). The baseline (resting) spleen volume was measured each minute during the last 5 min of the 20 min of rest before each test and was used as an average to compare with values obtained after apnea and exercise (Fig. 1). Spleen measurements were collected continuously for 5 min immediately following apnea and starting at 1 min following exercise. Spleen volume measurements were also made continuously between 20 and 25 min following the exercise bouts to obtain splenic recovery measurements (Fig. 1.).
Fig. 1Protocol overview of test day 1 and day 2. Arrows showing time points where blood samples were taken (for [Hb], serum albumin, and serum whole protein). Dashed lines show ultrasound measurements of the spleen; filled lines show measurements of \(}}\)O2, SpO2, and HR; numbers show number of minutes (‘) or seconds (‘’) of rest and conditions. Day 1 was always performed in the same order, whereas exercise conditions on day 2 were randomized in order. EX 55, EX 70, and EX85 = Exercise intensity corresponding to 55, 70, and 85% of \(}}\)O2max
Cardiorespiratory measurementsThroughout the apnea test, peripheral oxygen saturation (SpO2) and heart rate (HR) were continuously measured using a combined pulse oximeter (Medair LifeSense LS1-9R, Medair AB, Delsbo, Sweden) attached to the participant’s finger. Measurements of respiratory variables were made using an AMIS 2001 model C (Innovision A/S, Odense, Denmark) metabolic cart. Calibration of the metabolic carts flowmeter was performed with a 3-L syringe (Hans Rudolph, Kansas City, Missouri, USA) over a range from low to high flow rates and the gas analyzers were calibrated before each test with a gas mixture of 16.0% oxygen and 4.5% CO2 in N2 (Strandmöllen, Ljungby, Sweden). The ambient conditions were monitored before each test with an external device (Vaisala PTU 200, Vaisala Oy, Helsinki, Finland), and corrections were made to the internal ambient calibration of the metabolic cart. HR was monitored with a Polar V800 chest strap apparatus (Polar Electro Oy, Kempele, Finland). The resting periods were made sitting on a cushioned chair where all blood samples and spleen volume measurements were collected.
ProtocolApnea testThe apnea test consisted of three maximal voluntary static and dry apneas with 2 min of rest in between, which were performed in a sitting position following 25 min of sitting rest (Fig. 1). The participants were instructed to exhale completely, inhale one deep but not maximal breath and then to hold the breath as long as they could before voluntary termination (Schagatay et al. 2005). Through a pulse oximeter attached to the participant’s finger, a research assistant monitored the HR and SpO2. If the SpO2 fell below 60% at any point, the participants would be told to terminate the breath-hold and resume breathing. This never occurred. To avoid psychological effects, no feedback concerning apnea duration was provided to the participants.
\(\dot\) O 2max testThe roller skiing tests were all performed on a motor-driven roller skiing treadmill (Rodby Innovation AB, Vänge, Sweden), and Pro-Ski C2 (Sterners, Dala-Järna, Sweden) roller skis were equipped with Prolink (Salomon, Annecy, France) bindings. The participants wore a safety harness, during all tests, which was connected by a rope to an emergency brake. Following an additional 25 min of seated rest and sampling (Fig. 1.), a combined GE and \(\dot\)O2max test protocol was performed to establish the maximal aerobic capacity as well as for calculating roller skiing speeds for test day 2. The two-phased automated protocol started with a 5-minute constant speed GE measurement period and ended with a \(\dot\)O2max ramp time to exhaustion (TTE) test. The GE section was performed at 8 km·h−1 and after 5 min, the ramp part started and increased 0.5 km·h−1 each 30 s. All roller skiing tests were performed at a constant incline of seven degrees to ensure basis for a good diagonal skiing technique (Andersson et al. 2017). The test was terminated by an experienced test leader (HL) when the participant no longer could keep up with the speed of the treadmill.
Exercise intensitiesTo determine the effects of exercise intensity, three different submaximal exercise levels were performed in a randomized order during test day 2 (Fig. 1.). The participants commenced the test day as described in day 1, after which three exercise levels were performed. Roller skiing speeds were calculated according to a model based on the work by Andersson et al. (2017), to correspond to 55% (EX55), 70% (EX70), and 85% (EX85) of each participant’s individual \(\dot\)O2max from test day 1. All submaximal levels started with 4 min of warm-up at 55% intensity followed by 6 min at a randomized order. The low intensity was chosen to avoid the risk of an added sympathetic splenic response caused by starting at high intensity. Seated rest for 25 min followed each level of exercise. Blood samples were taken at 5 min before and 1 min after exercise, and spleen measurements were taken for 5 min prior to exercise as well as for 5 min starting 1 min after exercise. One minute was needed for the participant to get to the seated position after being assisted in taking off roller skis, poles, safety harness, and the mouthpiece from the metabolic cart.
AnalysisThe splenic diameter measurements obtained were used for volume calculations via the Pilström equation as follows:
\(V\)spleen = Lπ(WT-T2)/3 (Schagatay et al. 2005).
The splenic measurements were analyzed either as an average of five samples (baseline) or simply as the first spleen size following apnea and exercise. This method has recently been observed to have high reliability between repeated measurements (Holmström et al. 2022). Each condition is associated with a preceding baseline resting period, wherein splenic and hematological baseline values refer to measurements during this rest period prior to each condition separately. Individual splenic contraction was then calculated as an absolute (mL) and relative (%) change in the volume after the apnea and exercise from the associated baseline volume. Spleen volume was expressed in mL and in relation to body height (mL·cm−1).
[Hb], Hla, S-albumin, and S-protein samples were analyzed as one measurement within the recommended time for each sample type. \(\dot\)O2max was calculated as the average of the highest consecutive 30 s during the \(\dot\)O2max protocol. The criterion for reaching \(\dot\)O2max was a respiratory quotient (RQ) > 1.1, and Hla > 8 mmol·L−1. GE was calculated using treadmill speed and inclination, rolling resistance of roller skis, and metabolic data of \(\dot\)O2 and RER (Andersson et al. 2017).
Statistical analysisStatistical analyses were performed using IBM SPSS Statistics (Version 27.0; IBM Corporation, NY, US) with a significance level set to α < 0.05. Data were reported as mean (SD) and normal distribution of data was assessed using the Shapiro–Wilk test (p < 0.05). Repeated-measures two-way ANOVAs (within factors: condition; time) were used to identify if conditions (apnea; exercise intensities) influenced splenic contraction and [Hb]. Significant interactions were followed up with simple main effect analyses with one-way repeated-measures ANOVAs which were adjusted for multiple comparisons with Bonferroni adjustment. The magnitude of the effects was estimated with Cohen’s d effect size (ES), computed as the mean difference divided by the pooled SD. ESs are presented with 95% confidence intervals (CIs), and an ES of 0.2–0.3 was considered small, 0.4–0.7 as medium, and 0.8 as large (Lee 2016). Pearson’s product-moment correlation coefficients (r) were used to quantify associations between splenic contraction and [Hb] with XC skiing performance indicators (\(\dot\) O2max).
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