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We investigated the effects of recombinant human erythropoietin (rHuEPO) administration on exercise endurance, maximal aerobic performance and total hemoglobin mass (tHb). We hypothesized that frequent, small intravenous injections of epoetin β would increase time trial performance, peak oxygen uptake (V̇O2peak) and tHb in both males and females.We included 48 healthy, recreational to trained males (n = 24, mean ± standard deviation V̇O2peak of 55 ± 5 ml O2 × kg-1 × min-1) and females (n = 24; V̇O2peak of 46 ± 4 ml O2 × kg-1 × min-1) in a counter-balanced, double-blind, randomized, placebo-controlled study design stratified by sex. Time trial performance, V̇O2peak, and tHb were determined before and after intravenous injections of either rHuEPO (9 IU × kg bw-1 epoetin β) or saline (0.9% NaCl) three times weekly for 4 weeks.A time × treatment effect (P0.05) existed for time trial performance. Within the rHuEPO group, mean power output (MPO) increased by 4.1 ± 4.2% (P0.001). Likewise, a time × treatment effect (P0.001) existed for V̇O2peak, where the rHuEPO group improved V̇O2peak and peak aerobic power by 4.2 ± 6.1% (P0.001) and 2.9 ± 4.0% (P0.01), respectively. A time × treatment effect (P0.001) existed for tHb, where the rHuEPO group increased tHb by 6.7 ± 3.4% (P0.001). A main effect of 'sex' alone was also evident (P0.001) but no sex-specific interactions were found. No changes were observed in the placebo group for MPO, V̇O2peak, peak aerobic power or tHb.Micro-doses with intravenous rHuEPO provide a sufficient erythropoietic stimuli to augment tHb and enhance aerobic-dominated performance in both trained males and females.

Purpose: We investigated whether hepcidin and erythroferrone (ERFE) could complement the Athlete Biological Passport (ABP) in indirectly detecting a 130 mL packed red blood cells (RBCs) autologous blood transfusion. Endurance performance was evaluated.Methods: Forty-eight healthy men (n = 24) and women (n = 24) participated. Baseline samples were collected weekly followed by randomization to a blood transfusion (BT, n = 24) or control group (CON, n = 24). Only the BT group donated 450 mL whole blood from which 130 mL RBCs was reinfused four weeks later. Blood samples were collected 3, 7, 14, 21 and 28 days after donation, and 3, 6, and 24 hours and 2, 3, and 6 days following reinfusion. In the CON group samples were collected with the same frequency. Endurance performance was evaluated by a 650-kCal time trial (n = 13) before and one and six days after reinfusion.Results: A time×treatment effect existed (P < 0.05) for hepcidin and ERFE. Hepcidin was increased (P < 0.01) ~110 and 89% six and 24 hours after reinfusion. Using an individual approach (99% specificity, e.g. allowing 1:100 false-positive), sensitivities, i.e. true positives, of 30% and 61% was found for hepcidin and ERFE, respectively. For the ABP, the most sensitive marker was Off-hr score ([Hb] (g·L-1) - 60 × √RET%) (P < 0.05) with a maximal sensitivity of ~58% and ~ 9% following donation and reinfusion, respectively. Combining the findings for hepcidin, ERFE and the ABP yielded a sensitivity across all time-points of 83% following reinfusion in BT. Endurance performance increased 24 hours (+6.4%, P < 0.01) and six days following reinfusion (+5.8%, P < 0.01).Conclusions: Hepcidin and ERFE may serve as biomarkers in an anti-doping context following an ergogenic, small-volume blood transfusion.

Background: Indirect detection of recombinant human erythropoietin (rhEPO) abuse in Anti-Doping world is done by hematological module of athlete biological passport (ABP). However, actual markers (reticulocytes (%RET) and hemoglobin (HGB)) of this module are not enough specific and have difficulties to distinguish recombinant human erythropoietin (rhEPO) administration from altitude sojourn especially if both condition are merged. Previous study have demonstrated that erythropoiesis-related RNAs, genes 5’-aminolevulinate synthase 2 ( ALAS2) and carbonic anhydrase 1 ( CA1) in capillary dried blood spots (DBS) could be potential biomarkers to increased specificity and sensitivity of the ABP in case of hypoxic conditions. Study design and methods: In the present study, we monitored ALAS2 and CA1 expression in DBS after injection of 20 IU kg bw-1 epoetin alpha at sea level and the effect merged with altitude exposure. For this purpose, DBSs samples collected from 39 volunteers’ males and females were analyzed. Participants were divided in two groups (EPO and placebo) and subjects from both groups sojourned at sea level and at altitude. The purpose was to evaluate the performance of RNAs biomarkers for the detection of rHEPO injection in a longitudinal blinded setting. Results: Our results illustrated a clear significant increase of ALAS2 until to 300% when treated at sea level and at altitude. Regarding CA1 the increase is up to 200% in both conditions. Interestingly, our results suggested no combined effect of altitude and rhEPO injection on ALAS2 and CA1 biomarkers as previously demonstrated with %RET values. In placebo group. Moreover, no effect of altitude on ALAS2 and CA1 was observed in contrast to %RET parameter. Results from blind setting test demonstrated a performance of interpretation of 92% regarding ALAS2 and CA1 monitoring. Conclusion: Erythropoiesis-related RNAs in capillary DBS such as ALAS2 and CA1 were sensitive and specific biomarkers for low-dose rhEPO misuse at sea level and altitude. Interestingly, altitude seemed not to be a confounding factors for ALAS2 and CA1 biomarkers in contrast to %RET. Furthermore, this investigation provide evidence that low-dose of rhEPO injections can be detected in a longitudinal blinded setting with RNA biomarkers in DBS. World Anti-Doping Agency (WADA) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Blood doping is prohibited for athletes but has been a well-described practice within endurance sports throughout the years. With improved direct and indirect detection methods, the practice has allegedly moved towards micro-dosing, that is, reducing the blood doping regime amplitude. This narrative review evaluates whether blood doping, specifically recombinant human erythropoietin (rhEpo) treatment and blood transfusions are performance-enhancing, the responsible mechanism as well as detection possibilities with a special emphasis on micro-dosing. In general, studies evaluating micro-doses of blood doping are limited. However, in randomized, double-blinded, placebo-controlled trials, three studies find that infusing as little as 130 ml red blood cells or injecting 9 IU × kg bw

We evaluated whether or not changes in body composition following moderate hypoxic exposure for 4 weeks were different compared to sea level exposure.In a randomized crossover design, nine trained participants were exposed to 2320 m of altitude or sea level for 4 weeks, separated by 3 months. Body fat percentage (BF%), fat mass (FM), and fat-free mass (FFM) were determined before and after each condition by dual X-ray absorptiometry (DXA) and weekly by a bioelectrical impedance scanner to determine changes with a high resolution. Training volume was quantified during both interventions.Hypoxic exposure reduced (P 0.01) BF% by 2 ± 1 percentage points and increased (P 0.01) FFM by 2 ± 2% determined by DXA. A tending time × treatment effect existed for FM determined by DXA (P = 0.06), indicating a reduced FM in hypoxia by 8 ± 7% (P 0.01). Regional body analysis revealed reduced (P 0.01) BF% and FFM and an increased (P 0.01) FFM in the truncus area. No changes were observed following sea level. Bioelectrical impedance determined that BF%, FM, and FFM did not reveal any differences between interventions. Urine specific gravity measured simultaneously as body composition was identical. Training volume was similar between interventions (509 ± 70 min/week vs. 432 ± 70 min/week, respectively).Four weeks of altitude exposure reduced BF% and increased FFM in trained individuals as opposed to sea level exposure. The results also indicate that a decrease in FM is greater at altitude compared to sea level. Changes were specifically observed in the truncus area.

Current markers of iron deficiency (ID) such as ferritin and hemoglobin have shortcomings, and hepcidin and erythroferrone (ERFE) could be of clinical relevance in relation to early assessment of ID. Here, we evaluate whether exposure to altitude-induced hypoxia (2,320 m) alone, or in combination with recombinant human erythropoietin (rHuEPO) treatment, affects hepcidin and ERFE levels before alterations in routine ID biomarkers and stress erythropoiesis manifest. Two interventions were completed, each comprising a 4-wk baseline, a 4-wk intervention at either sea level or altitude, and a 4-wk follow-up. Participants (n=39) were randomly assigned to 20 IU·kg body wt-1 rHuEPO or placebo injections every second day for 3 wk during the two intervention periods. Venous blood was collected weekly. Altitude increased ERFE (P ≤ 0.001) with no changes in hepcidin or routine iron biomarkers, making ERFE of clinical relevance as an early marker of moderate hypoxia. rHuEPO treatment at sea level induced a similar pattern of changes in ERFE (P < 0.05) and hepcidin levels (P < 0.05), demonstrating the impact of accelerated erythropoiesis and not of other hypoxia-induced mechanisms. Compared to altitude alone, concurrent rHuEPO treatment and altitude exposure induced additive changes in hepcidin (P < 0.05) and ERFE (P ≤ 0.001) parallel with increases in hematocrit (P < 0.001), demonstrating a relevant range of both hepcidin and ERFE. A poor but significant correlation between hepcidin and ERFE was found (R2 = 0.13, P < 0.001). The findings demonstrate that hepcidin and ERFE are more rapid biomarkers of changes in iron demands than routine iron markers. Finally, ERFE and hepcidin may be sensitive markers in an anti-doping context.Clinical trial registration: Clinical trials identifier, NCT04227665

Some have questioned the evidence for performance-enhancing effects of several substances included on the World Anti-Doping Agency's Prohibited List due to the divergent or inconclusive findings in randomized controlled trials (RCTs). However, inductive statistical inference based on RCTs-only may result in biased conclusions because of the scarcity of studies, inter-study heterogeneity, too few outcome events, or insufficient power. An abductive inference approach, where the body of evidence is evaluated beyond considerations of statistical significance, may serve as a tool to assess the plausibility of performance-enhancing effects of substances by also considering observations and facts not solely obtained from RCTs. Herein, we explored the applicability of an abductive inference approach as a tool to assess the performance-enhancing effects of substances included on the Prohibited List. We applied an abductive inference approach to make inferences on debated issues pertaining to the ergogenic effects of recombinant human erythropoietin (rHuEPO), beta2-agonists and anabolic androgenic steroids (AAS), and extended the approach to more controversial drug classes where RCTs are limited. We report that an abductive inference approach is a useful tool to assess the ergogenic effect of substances included on the Prohibited List - particularly for substances where inductive inference is inconclusive. Specifically, a systematic abductive inference approach can aid researchers in assessing the effects of doping substances, either by leading to suggestions of causal relationships or identifying the need for additional research.

We investigated whether immature reticulocyte fraction (IRF) and immature reticulocytes to red blood cells ratio (IR/RBC) are sensitive biomarkers for low-dose recombinant human erythropoietin (rhEpo) treatment at sea-level (SL) and moderate altitude (AL) and whether multi (FACS) or single (Sysmex-XN) fluorescence flow cytometry is superior for IRF and IR/RBC determination. Thirty-nine participants completed two interventions, each containing a four-week baseline, a four-week SL or AL (2,230m) exposure and a four-week follow-up. During exposure, rhEpo (20 IU·kg-1) or placebo (PLA) was injected at SL (SLrhEpo n = 25, SLPLA n = 9) and AL (ALrhEpo n = 12, ALPLA n = 27) every second day for three weeks. Venous blood was collected weekly. Sysmex measurements revealed that IRF and IR/RBC was up to ~70% (P < 0.01) and ~190% (P < 0.001) higher in SLrhEpo than SLPLA during treatment and up to ~45% (P < 0.001) and ~55% (P < 0.01) lower post-treatment, respectively. Compared with ALPLA, IRF and IR/RBC was up to ~20% (P < 0.05) and ~45% (P < 0.001) lower post-treatment in SLrhEpo, respectively. In ALrhEpo, IRF and IR/RBC was up to ~40% (P < 0.05) and ~110% (P < 0.001) higher during treatment and up to ~25% (P < 0.05) and ~40% (P < 0.05) lower post-treatment, respectively, compared with ALPLA. Calculated thresholds provided ~90% sensitivity for both biomarkers at SL and 33% (IRF) and 66% (IR/RBC) at AL. Specificity was >99%. Single-fluorescence flow cytometry coefficient of variation was >twofold higher at baseline (P < 0.001), and provided larger or similar changes compared to multi-fluorescence, albeit with smaller precision. In conclusion, IRF and IR/RBC were sensitive and specific biomarkers for low-dose rhEpo misuse at SL and AL.

Objectives Optimized concurrent training regimes are warranted in physical training of military-, law enforcement- and rescue-personnel. This study investigated if four 15-min endurance training sessions weekly improve aerobic capacity and performance more than one 60-min endurance session weekly during the initial phase of a Basic Military Training program. Design A randomized training intervention study with functional and physiological tests before and after the intervention. Methods Military conscripts (n = 290) were randomly allocated to three groups completing 9 weeks training. Weekly training consisted of four endurance and four strength training sessions lasting 15 min each (‘Micro-training’: MIC); one strength and one endurance session lasting 60 min each (‘Classical-training’: CLA) or two 60 min sessions of standard military training (‘Control-training’: CON). Results Both 12-min (∼7−10%) and shuttle run performance (∼35−42%) improved (P ≤ 0.001) similarly in all groups. Likewise, functional 2-min maximal repetition exercise capacity increased (P ≤ 0.05) similarly in all groups (Lunges ∼17−24 %; PushUp ∼10−20%; AbdominalFlexions∼21−23%). Peak oxygen uptake changes depended on group (P ≤ 0.05) with increases (P ≤ 0.01) in MIC (7 ± 7%, n = 23) and CON (12 ± 18%, n = 17) and no changes in CLA. Maximal m. vastus lateralis citrate synthase activity decreased 14 ± 26% (P ≤ 0.001, n = 18) in CLA. Likewise, maximal m. vastus lateralis 3-hydroxyacyl-CoA dehydrogenase activity decreased 8 ± 17% in MIC (n = 28) and 14 ± 24% in CLA (n = 18). Conclusions Four 15-min endurance training sessions weekly improves running performance and strength-endurance similarly to one 60 min session. Peak oxygen uptake only increases with more than one endurance session weekly and leg muscle oxidative capacity appears reduced after basic military training.

We examined whether a semi-automated carbon monoxide (CO) rebreathing method accurately detect changes in blood volume (BV) and total hemoglobin mass (tHb). Furthermore, we investigated whether a supine position with legs raised reduced systemic CO dilution time, potentially allowing a shorter rebreathing period. Nineteen young healthy males participated. BV and tHb was quantified by a 10-min CO-rebreathing period in a supine position with legs raised before and immediately after a 900 ml phlebotomy and before and after a 900 ml autologous blood reinfusion on the same day in 16 subjects. During the first CO-rebreathing, arterial and venous blood samples were drawn every 2 min during the procedure to determine systemic CO equilibrium in all subjects. Phlebotomy decreased (P

Angiotensin-converting enzyme (ACE) inhibitor treatment is widely applied, but the fact that plasma ACE activity is a potential determinant of training-induced local muscular adaptability is often neglected. Thus, we investigated the hypothesis that ACE inhibition modulates the response to systematic aerobic exercise training on leg and arm muscular adaptations.Healthy, untrained, middle-aged participants (40 ± 7 yrs) completed a randomized, double-blinded, placebo-controlled trial. Participants were randomized to placebo (PLA: CaCOForty-eight participants (nEight weeks of high-intensity whole-body exercise training improves markers of skeletal muscle mitochondrial oxidative capacity, glycolytic capacity and angiogenesis, with no overall effect of pharmacological ACE inhibition in healthy adults.

Objectives: To investigate whether recombinant human erythropoietin (rHuEPO) injections during an altitude training camp impact heart function. Methods: Thirty (12 women) moderately trained subjects stayed at 2320 m altitude for 4 weeks while training. Subjects were randomized to placebo (isotonic saline) or rHuEPO (20 IU/kg body weight) i.v. injections. Transthoracic echocardiography imaging was acquired 3 days after arrival to altitude and prior to the first placebo or rHuEPO injection as well as one day after the last rHuEPO injection three weeks later. Results: rHuEPO did not alter cardiovascular morphology parameters, systolic or diastolic function. In the placebo group, altitude exposure improved left ventricle (LV) systolic function due to an increased twist angle but rHuEPO had no additional effects. Pulmonary arterial systolic pressure was unaffected in either group. Notably, rHuEPO hampered LV untwist rate without affecting LV early filling. Conclusion: rHuEPO provided during mild altitude exposure does not cause any major effects on heart function. The observed alteration in LV untwist induced by rHuEPO is unlikely to have a meaningful clinical effect., World Anti-Doping Agency ISF17R02NN

This study evaluated whether recombinant human erythropoietin (rhEpo) treatment combined with chronic hypoxia provided an additive erythropoietic response and whether the Athlete Biological Passport (ABP) sensitivity improved with hypoxia. Two interventions were completed, each containing four weeks baseline, four weeks exposure at sea-level or 2,320m of altitude and four weeks follow-up. Participants were randomly assigned to 20 IU·kg bw-1 rhEpo or placebo injections every second day for three weeks during the exposure period at sea-level (rhEpo n=25, placebo n=9) or at altitude (rhEpo n=12, placebo n=27). Venous blood was analyzed weekly. Combining rhEpo and hypoxia induced larger changes compared with rhEpo or hypoxia alone for [Hb] (p < 0.001, p > 0.05, respectively), reticulocyte percentage (p < 0.001) and OFF-hr score (p < 0.01, p < 0.001, respectively). The most pronounced effect was observed for reticulocyte percentage with up to ~35% (p < 0.001) and ~45% (p < 0.001) higher levels compared with rhEpo or hypoxia only, respectively. The ABP sensitivity for the combined treatment was 54 and 35 percentage points higher for [Hb] (p < 0.05) and reticulocyte percentage (p < 0.05), respectively, but similar for OFF-hr score, compared with rhEpo at sea-level. Across any time point, [Hb] and OFF-hr score combined identified 14 unique true-positive participants (56%) at sea-level and 12 unique true-positive participants (100%) at altitude. However, a concurrent reduction in specificity existed at altitude. In conclusion, rhEpo treatment combined with hypoxic exposure provided an additive erythropoietic response compared to rhEpo or hypoxic exposure alone. Correspondingly, ABP was more sensitive to rhEpo at altitude than at sea-level, but a compromised specificity existed with hypoxic exposure.

Erythroid terminal differentiation entails cell divisions that are coupled to progressive decreases in cell size. EpoR signaling is essential for the survival of erythroid precursors, but it is unclear whether it has other functions in these cells. Here we endowed mouse precursors that lack the EpoR with survival signaling, finding that this was sufficient to support their differentiation into enucleated red cells, but that the process was abnormal. Precursors underwent fewer and slower cell cycles and yet differentiated into smaller red cells. Surprisingly, EpoR further accelerated cycling of early erythroblasts, the fastest cycling cells in the bone marrow, while simultaneously increasing their cell size. EpoR-mediated formation of larger red cells was independent of the established pathway regulating red cell size by iron through Heme-regulated eIF2α kinase (HRI). We confirmed the effect of Epo on red cell size in human volunteers, whose mean corpuscular volume (MCV) increased following Epo administration. This increase persisted after Epo declined and was not the result of increased reticulocytes. Our work reveals a unique effect of EpoR signaling on the interaction between the cell cycle and cell growth. Further, it suggests new diagnostic interpretations for increased red cell volume, as reflecting high Epo and erythropoietic stress.

Objectives To evaluate whether the donation of 900 mL of blood reduces the central blood volume (CBV) assessed by thoracic electrical impedance (TI) and plasma pro-atrial natriuretic peptide (proANP). Background Donation of 450 mL of blood carries a 1% risk of a vasovagal reaction. Withdrawal of 900 mL of blood decreases cardiac output; however, the effect on CBV remains unknown. Methods/materials A randomised, single-blinded, placebo-controlled, crossover design was used, where 21 healthy semi-recumbent men donated 2 × 450 mL blood or were sham-phlebotomised. Changes in CBV were estimated by proANP and TI at 1.5 (TI1.5 ) and 100 (TI100 ) kHz, reflecting extracellular volume and (regional) total body water, respectively, and the index value (IDX; 1/T1.5 -1/TI100 ) was used to estimate changes in intracellular (red cell) volume. Systolic, diastolic and mean arterial blood pressure; heart rate; stroke volume; cardiac output; and systemic vascular resistance were monitored. After completion of the study, 1000 mL of isotonic saline was infused. Results Changes (mean% ± SD) in TI1.5 , TI100 and IDX were similar after 450 mL (-0.2 ± 1.6%, 0.0 ± 1.1%, -0.4 ± 10.1%) and 900 mL (0.1 ± 1.6%, 0.2 ± 1.5% and -2.0 ± 15.8%) of blood donation compared to after a sham donation of 450 mL (-0.9 ± 1.2%, -0.5 ± 1.5% and -0.1 ± 6.1%) and 900 mL (-1.2 ± 1.5%, -0.6 ± 1.3% and 0.5 ± 9.9%). In addition, changes in plasma proANP were similar after 450 and 900 mL of blood donation (-0.8 ± 6.7% and -7.6 ± 7.9%) as after sham donations (1.3 ± 7.3% and -4.5 ± 5.6%). Monitoring haemodynamic variables revealed that stroke volume decreased after the donation of 900 mL of blood (-12 ± 12 mL) compared to sham donations. Conclusion During a 900-mL blood loss in semi-recumbent men, CBV measured by TI and plasma proANP is not affected.

Detailed physiological phenotyping was hypothesized to have predictive value for Olympic distance cross-country mountain bike (XCO-MTB) performance. Additionally, mean (MPO) and peak power output (PPO) in 4 × 30 s all-out sprinting separated by 1 min was hypothesized as a simple measure with predictive value for XCO-MTB performance. Parameters indicative of body composition, cardiovascular function, power and strength were determined and related to XCO-MTB national championship performance (n = 11). Multiple linear regression demonstrated 98% of the variance (P < 0.001) in XCO-MTB performance (tXCO-MTB; [min]) is explained by maximal oxygen uptake relative to body mass (VO2peak,rel; [ml/kg/min]), 30 s all-out fatigue resistance (FI; [%]) and with a minor contribution from quadriceps femoris maximal torque (Tmax; [Nm]): tXCO-MTB = -0.217× VO2peak,rel.-0.201× FI+ 0.012× Tmax+ 85.4. Parameters with no additional predictive value included hemoglobin mass, leg peak blood flow, femoral artery diameter, knee-extensor peak workload, jump height, quadriceps femoris maximal voluntary contraction force and rate of force development. Additionally, multiple linear regression demonstrated parameters obtained from 4x30s repeated sprinting explained 88% of XCO-MTB variance (P < 0.001) with tXCO-MTB = -5.7× MPO+ 5.0× PPO+ 55.9. In conclusion, XCO-MTB performance is predictable from VO2peak,rel and 30 s all-out fatigue resistance. Additionally, power variables from a repeated sprint test provides a cost-effective way of monitoring athletes XCO-MTB performance.

The novel hypothesis that "Live High-Train Low" (LHTL) does not improve sport-specific exercise performance (e.g., time trial) is discussed. Indeed, many studies demonstrate improved performance after LHTL but, unfortunately, control groups are often lacking, leaving open the possibility of training camp effects. Importantly, when control groups, blinding procedures, and strict scientific evaluation criteria are applied, LHTL has no detectable effect on performance.

The erythroid terminal differentiation program couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. Here we use Epor−/− mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. We find that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. EpoR-regulation of cell size is independent of established red cell size regulation by iron. High erythropoietin (Epo) increases red cell size in wild-type mice and in human volunteers. The increase in mean corpuscular volume (MCV) outlasts the duration of Epo treatment and is not the result of increased reticulocyte number. Our work shows that EpoR signaling alters the relationship between cycling and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress., Maturing erythroblasts become smaller with every cell division. Here, the authors show that Epo stimulation promotes cell division and also generates larger red cells, and that this occurs in mouse and human cells, suggesting that red cell size could be a diagnostic marker for hypoxic stress.

The aim was to investigate whether 6 weeks of normobaric “Live High-Train Low” (LHTL) using altitude tents affect highly trained athletes incremental peak power, 26-km time-trial cycling performance, 3-min all-out performance, and 30-s repeated sprint ability. In a double-blinded, placebo-controlled cross-over design, seven highly trained triathletes were exposed to 6 weeks of normobaric hypoxia (LHTL) and normoxia (placebo) for 8 h/day. LHTL exposure consisted of 2 weeks at 2500 m, 2 weeks at 3000 m, and 2 weeks at 3500 m. Power output during an incremental test, ~26-km time trial, 3-min all-out exercise, and 8 × 30 s of all-out sprint was evaluated before and after the intervention. Following at least 8 weeks of wash-out, the subjects crossed over and repeated the procedure. Incremental peak power output was similar after both interventions [LHTL: 375 ± 74 vs. 369 ± 70 W (pre-vs-post), placebo: 385 ± 60 vs. 364 ± 79 W (pre-vs-post)]. Likewise, mean power output was similar between treatments as well as before and after each intervention for time trial [LHTL: 257 ± 49 vs. 254 ± 54 W (pre-vs-post), placebo: 267 ± 57 vs. 267 ± 52 W (pre-vs-post)], and 3-min all-out [LHTL: 366 ± 68 vs. 369 ± 72 W (pre-vs-post), placebo: 365 ± 66 vs. 355 ± 71 W (pre-vs-post)]. Furthermore, peak- and mean power output during repeated sprint exercise was similar between groups at all time points (n = 5). In conclusion, 6 weeks of normobaric LHTL using altitude tents simulating altitudes of 2500–3500 m conducted in a double-blinded, placebo-controlled cross-over design do not affect power output during an incremental test, a ~26-km time-trial test, or 3-min all-out exercise in highly trained triathletes. Furthermore, 30 s of repeated sprint ability was unaltered.

Recombinant human erythropoietin (rhEpo) can improve human performance, but misuse remains difficult to detect. C-terminal fibroblast growth factor 23 (cFGF23) was recently demonstrated to increase following injection of a single high dose rhEpo, but the effect of more frequent low doses is unknown. Using a randomized double-blind placebo-controlled design, we investigated whether 2 weeks of subcutaneous injections three times a week of 50 IU/kg Eprex (low-dose) or 20 IU/kg Eprex (micro-dose) increase cFGF23 levels compared with saline (placebo) injections in 24 healthy males. Venous blood was sampled at day -3, 0, 1, 3, 11, 14, 18, and 25 of the treatment and analyzed for cFGF23 and erythropoietin concentration ([EPO]). The level of cFGF23 was similar at days -3, 0, 1, 3, 11, 14, 18, and 25 with the low-dose (23 ± 4, 26 ± 5, 23 ± 7, 27 ± 6, 25 ± 8, 24 ± 10, 22 ± 5, and 24 ± 7 RU/mL, respectively), micro-dose (23 ± 6, 25 ± 5, 23 ± 8, 28 ± 9, 27 ± 7, 25 ± 9, 25 ± 5, and 23 ± 6 RU/mL, respectively) and placebo (23 ± 6, 24 ± 6, 26 ± 7, 26 ± 6, 31 ± 6, 31 ± 7, 24 ± 4, and 27 ± 8 RU/mL, respectively) treatment. The correlation coefficient between plasma [EPO] and plasma cFGF23 levels was R2 = 0.01 and insignificant. The results demonstrate that cFGF23 is not sensitive to low doses of subcutaneous rhEpo injections in healthy males.

Purpose: To investigate the hypothesis that a therapeutic oral dose of Tramadol improves cycling time trial performance and compromises motor-cognitive performance in highly trained cyclists.Methods: Following two familiarization trials, 16 highly trained cyclists completed a preloaded time trial (1 hour at 60 % of peak power followed by a 15 km time trial) after ingestion of 100 mg Tramadol or placebo in a double-blind placebo-controlled counterbalanced cross-over design separated by at least 4 days wash-out. Visuo-motor tracking and math tasks were completed during the preload (n=10) to evaluate effects on cognition and fine motor performance.Results: Time trial mean power output (298±42 W vs. 294±44 W) and performance (1474±77 s vs. 1483±85 s) were similar with Tramadol and placebo treatment, respectively. In addition, there were no differences in perceived exertion, reported pain, blood pH, lactate or HCO3 concentrations across trials. Heart rate was higher (PPConclusion: In highly trained cyclists, ingestion of 100 mg Tramadol does not improve performance in a 15 km cycling time trial that was completed after a one hour preload at 60 % peak power. Additionally, a therapeutic dose of Tramadol does not compromise complex motor-cognitive or simple fine motor performances.

Objectives Military-, rescue- and law-enforcement personnel require a high physical capacity including muscular strength. The present study hypothesized that 9 weeks of volume matched concurrent short frequent training sessions increases strength more efficiently than less frequent longer training sessions. Design A randomized training intervention study with functional and physiological tests before and after the intervention. Methods Military conscripts (n = 290) were assigned to micro-training (four 15-min strength and four 15-min endurance bouts weekly); classical-training (one 60-min strength and one 60-min endurance training session weekly) or a control-group (two 60-min standard military physical training sessions weekly). Results There were no group difference between micro-training and classical-training in measures of strength. Standing long jump remained similar while shotput performance was reduced (P ≤ 0.001) in all three groups. Pull-up performance increased (P ≤ 0.001) in micro-training (7.4 ± 4.6 vs. 8.5 ± 4.0 repetitions, n = 59) and classical-training (5.7 ± 4.1 vs. 7.1 ± 4.2 repetitions, n = 50). Knee extensor MVC increased (P ≤ 0.01) in all groups (micro-training, n = 30, 11.5 ± 8.9%; classical-training, n = 24, 8.3 ± 11.5% and control, n = 19, 7.5 ± 11.8%) while elbow flexor and hand grip MVC remained similar. Micro-training increased (P ≤ 0.05) type IIa percentage from 32.5 ± 11.0% to 37.6 ± 12.3% (n = 20) and control-group increased (P ≤ 0.01) type IIax from 4.4 ± 3.0% to 11.6 ± 7.9% (n = 8). In control-group type I, fiber size increased (P ≤ 0.05) from 5121 ± 959 μm to 6481 ± 2084 μm (n = 5). Satellite cell content remained similar in all groups. Conclusions Weekly distribution of low-volume concurrent training completed as either eight 15-min bouts or two 60-min sessions of which 50% was strength training did not impact strength gains in a real-world setting.

The time course of plasma volume (PV) reduction following an increased training load period is unknown and was investigated. The accompanying fluctuations in [Hb] and OFF-hr score were analyzed in the Athlete Biological Passport. Further, whether fluctuations in plasma albumin, soluble transferrin receptors (sTfR), and pro-atrial natriuretic peptide (proANP) concentrations correlate with PV fluctuations was investigated. Eleven high-level competitive cyclists were investigated for 3 weeks. After initial measurements in week 1, training load was increased ~250% in week 2 followed by a reversion to baseline training load in week 3. PV and hematological variables were determined frequently during all weeks. The higher training load in week 2 increased (P

To minimize the chances of being caught after doping with recombinant human erythropoietins (rhEPO), athletes have turned to new practices using micro-doses and excess fluid ingestion to accelerate elimination and decrease the probability of detection. Our objective was to test the sensitivity of detection by validated methods (IEF: isoelectric focusing; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis) when such practices are used. First, after a three-week rhEPO boost period and 10 days of wash out, detection of a single 900 IU micro-dose of Eprex® was evaluated in healthy male subjects. After an injection in the evening, urine and plasma samples were collected the following morning. Half of the subjects then drank a bolus of water and new samples were collected 80 min later. Interestingly, rhEPO was detected in 100% of the samples even after water ingestion. A second similar protocol was then performed with a single injection of a micro-dose of rhEPO (500 IU or 900 IU), without a prior rhEPO boost. In addition, urine and plasma samples were also collected 15 and 20 h post rhEPO administration. Once again drinking water did not affect the rate of detection. Urine appeared a better matrix to detect micro-doses after 10 h, enabling between 92% and 100% of identification at that time. The rate of identification decreased rapidly thereafter, in particular for the 500 IU micro-dose. However IEF analysis still resulted in 71% identification of rhEPO in urine after 20 h. These results could help to define a better strategy for controlling and identifying athletes using rhEPO micro-doses. Copyright © 2016 John Wiley & Sons, Ltd.

EpoR signaling is essential for the survival of erythroid precursors during terminal differentiation, when these cells undergo 3-5 cell divisions that are coupled to a rapid loss in cell size. The absolute dependence of erythroblasts on EpoR signaling for survival makes it challenging to identify other essential functions of EpoR in these cells. Key open questions include a role for EpoR in cell cycle regulation; and a potential role for EpoR in the induction of erythroid genes. To address these gaps, we developed a genetic system that allowed us to examine essential non-survival functions of EpoR signaling in erythroid differentiation. We rescued mouse Epor-/- fetal liver erythroid progenitors from apoptosis by transduction with the anti-apoptotic protein bcl-xL, comparing their ensuing differentiation with that of Epor-/- progenitors that were rescued by re-introduction of the EpoR. We found that the bcl-xL survival signal, in the absence of any EpoR signaling, was sufficient to allow erythroid terminal differentiation and formation of anucleated red cells. However, key features were abnormal. First, erythroblasts underwent slower and fewer cell cycles, generating fewer than half the number of red cells, showing an essential role for EpoR signaling in the regulation of cell cycle speed and number. Second, we found that EpoR signaling unexpectedly increased red cell size, in spite of the concurrent increase in the number of cell divisions. Using mice doubly deleted for both EpoR and HRI, we found that EpoR regulation of red cell size is independent of the well-described red cell size regulation by iron and heme. Healthy human volunteers that were administered Epo showed an increase in mean corpuscular volume (MCV) that persisted long after Epo and reticulocyte levels returned to baseline, supporting a role for the regulation of red cell size by Epo and suggesting new diagnostic interpretations for this common clinical test. Together, our findings reveal novel functions for the EpoR in erythroid differentiation including an unexpected role in the regulation of red cell size.

Purpose: This study tested the hypothesis that autologous blood transfusion (ABT) of ~50% of the red blood cells (RBCs) from a standard 450 ml phlebotomy would increase mean power in a cycling time trial. Additionally, the study investigated whether further ABT of RBCs obtained from another 450 ml phlebotomy would increase repeated cycling sprint ability.Methods: In a randomized, double-blind, placebo-controlled crossover design (3-month wash-out), nine highly trained male subjects donated two 450 ml blood bags each (BT-trial) or were sham phlebotomized (PLA-trial). Four weeks later, a 650 kcal time trial (n=7) was performed three days before and 2 h after receiving either ~50% (135 ml) of the RBCs or a sham transfusion. On the following day, transfusion of RBCs (235 ml) from the second donation or sham transfusion was completed. A 4×30 s all-out cycling sprint interspersed by 4 min of recovery was performed six days before and three days after the second ABT (n=9).Results: The mean power was increased in time trials from before to after transfusion (PConclusion: ABT of only ~135 ml of RBCs is sufficient to increase mean power in a 650 kcal cycling time trial by ~5% in highly trained men. In contrast, a combined high-volume transfusion of ~135 and ~235 ml of RBCs does not alter 4×30 s all-out cycling performance interspersed with 4 min of recovery.

The sensitivity of the adaptive model of the Athlete Biological Passport (ABP) and reticulocyte percentage (ret%) in detection of recombinant human erythropoietin (rHuEPO) misuse was evaluated using both a long-term normal dose and a brief high dose treatment regime. Sixteen subjects received either 65 IU rHuEPO × kg-1 every second day for two weeks (normal-dose), 390 IU rHuEPO × kg-1 on three consecutive days (high-dose), or frequent placebo treatment for 13 days in a randomized, placebo-controlled, double-blind crossover design. Blood variables were measured 4, 11, and 25 days following treatment initiation. The ABP based on haemoglobin concentration ([Hb]) and OFF-hr score ([Hb] - 60 × √ret%) yielded atypical profiles following both normal-dose and high-dose treatment (0 %, 31 %, 13 % vs. 21 %, 33 %, 20 % at days 4, 11, and 25 after normal and high dose, respectively). Including ret% as a stand-alone marker for atypical blood profiles increased (P < 0.05) the sensitivity of the adaptive model at day 11 to 63 % and 67 % for normal-dose and high-dose rHuEPO administration, respectively. In conclusion, ~30 % of subjects injecting a normal-dose rHuEPO for two weeks or a high-dose rHuEPO for three days will present an atypical ABP profile. Including ret% as a stand-alone parameter improves the sensitivity two-fold. Copyright © 2015 John Wiley & Sons, Ltd.

The impact of altitude training on haematological parameters and the Athlete Biological Passport (ABP) was evaluated in international-level elite athletes. One group of swimmers lived high and trained high (LHTH, n = 10) for three to four weeks at 2130 m or higher whereas a control group (n = 10) completed a three-week training camp at sea-level. Haematological parameters were determined weekly three times before and four times after the training camps. ABP thresholds for haemoglobin concentration ([Hb]), reticulocyte percentage (RET%), OFF score and the abnormal blood profile score (ABPS) were calculated using the Bayesian model. After altitude training, six swimmers exceeded the 99% ABP thresholds: two swimmers exceeded the OFF score thresholds at day +7; one swimmer exceeded the OFF score threshold at day +28; one swimmer exceeded the threshold for RET% at day +14; and one swimmer surpassed the ABPS threshold at day +14. In the control group, no values exceeded the individual ABP reference range. In conclusion, LHTH induces haematological changes in Olympic-level elite athletes which can exceed the individually generated references in the ABP. Training at altitude should be considered a confounding factor for ABP interpretation for up to four weeks after altitude exposure but does not consistently cause abnormal values in the ABP.