miRNA Isolation

Sample amounts were standardized by volume: The same volume of plasma was used for each RNA isolation, and the same volume of purified RNA was used for all further analyses. The miRNAs were isolated from 50 μL (qRT-PCR) or 200 μL (PANEL screen) of plasma using a column-based protocol (miRNeasy Serum/Plasma Advanced Kit, (Qiagen, Hilden, Germany) according to the manufacturer's protocol. cel-miR-39 from *Caenorhabditis elegans* (1 nM) was spiked in. In the final step, total RNA (>18 nucleotides) was eluted using 20 μL of RNase-free water.

### Reverse Transcription and miRNA Profiling

For reverse transcription, the miRCURY LNA RT Kit (Qiagen, Hilden, Germany) was used. Undiluted complementary DNA (cDNA, 20 μL) was used for miRCURY LNA miRNA Focus Panel Human Serum/Plasma (YAHS-106Y) in the 2 × 96-well plate format. The Human Serum/Plasma Focus Panel includes 179 miRNA assays targeting relevant miRNAs, reference miRNAs, and spike-in controls.

### Reverse Transcription and qRT-PCR

Following reverse transcription as described above, quantitative real-time PCR was performed using miRCURY LNA miRNA PCR assays (Appendix A) in a 10-μL reaction containing 3 μL of cDNA (1:30) and a CFX real-time PCR detection system (BioRad, Munich, Germany). Assays were performed in triplicate. The amount of the respective miRNA was normalized to miR-425-3p and cel-miR-39.

For the miRCURY miRNA PCR analysis, v1.0 raw *C*t data from real-time PCR were up-loaded at (https://www.qiagen.com/us/shop/genes-and-pathways/data-analysis-center-overviewpage). Cel-miR-39-3p was used as an internal spike-in amplification control. A *C*t cut-off of 35 was set as the lower limit of detection. A global *C*t mean of expressed miRNAs was used for normalization, and the fold change was calculated as (2−ΔΔ*<sup>C</sup>*t), which represents the average normalized miRNA expression (2−ΔΔ*<sup>C</sup>*t) of the samples in the test group divided by the average normalized miRNA expression (2−ΔΔ*<sup>C</sup>*t) of the samples in the control group.

### 4.7.2. Self-Reported Outcomes

Self-reported outcomes consisted of rates of perceived exertion (RPE-Borg; Likert 6 to 20 point scale) [45], current well-being assessments (feeling scale: (+5 to −5, 10 point Likert scale)), and fatigue reporting (numeric rating scale NRS: 0 to 10 points). All self-reported parameters were assessed once after each intervention. The participants were asked to refer to the highest intensity during (RPE and feeling scale) or at the end (fatigue) of each intervention.

### *4.8. Data Analyses and Statistics*

For all outcomes assessed before and after each intervention, real values and absolute pre-to-post differences were used for further analysis. Continuously assessed variables were processed in their real values.

After the following plausibility control, all analyses were performed based on the results of the initial checking for relevant underlying assumptions to test for parametric or nonparametric characteristics (data, distribution of the variances and variance homogeneity). Between-group differences and pre-to-post changes were assessed using omnibus and follow-up post-hoc testing. SPSS 23 (SPSS Inc., Chicago, IL, USA) and GraphPad software PRISM5 for Mac (GraphPad Software, La Jolla, CA, USA) were used to conduct all statistical calculations and create figures. An alpha-error level of 5% was considered to be a relevant cut-off value for significance testing, with *p*-values below 0.05 indicating significant differences.

Friedman tests were performed for omnibus between-group comparisons for all resistance training outcomes (or the a priori calculated differences). For significant omnibus testing, post-hoc comparisons using post-hoc Bonferroni–Holm tests and alpha-error-adjusted Mann–Whitney-U-tests were performed. For pre-to-post significance testing, Wilcoxon tests were performed.

To identify significant miRNA expression changes between conditions, a fold regulation was calculated, and a fold-change threshold of 1.5 was defined. Significant miRNA expression changes were visualized using the volcano plot. For each miRNA showing a significant expression change, a pairwise group comparison (Student's *t*-test) was made based on the 2−ΔΔ*C*<sup>t</sup> value of the replicate samples. The *p*-value calculation was based on a parametric, two-sample, equal variance, unpaired, and two-tailed distribution.

The potential associations between the kinematic (treatment) e ffects of the miRNA and lactate were analyzed using partial linear regression with the covariate group allocation.

**Author Contributions:** J.V., K.T., T.E. and L.V. designed the experiments. J.V., K.T., T.E., D.N. and L.V. interpreted the findings. J.V. and K.T. wrote the first draft of the manuscript. J.V., K.T., C.T. and D.N. analyzed and performed the experiments. D.N. performed statistical analyses. C.T., T.S.-R. and W.B. discussed the results and revised the manuscript. All authors edited and approved the manuscript.

**Funding:** This research was funded by the German Research Foundation (Bonn, Germany, TR 1137/2-1 to K.T) and the Anna-Maria and Uwe-Karsten Kühl Foundation (T188/30462/2017, Bad Nauheim, Germany to K.T.).

**Acknowledgments:** The authors thank Monika Rieschel for excellent technical assistance, Jan Wilke for excellent support in the preparation of the study design, Andres Rosenhagen for excellent technical support in the ultrasonography. Elizabeth Martinson of the KHFI Editorial O ffice provided editorial assistance during preparation of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
