*2.3. Basic Resistance Training Outcomes*

### 2.3.1. Objective Outcomes of the Training Interventions

Lactate concentration and heart rate were increased after all training interventions (*p* < 0.001) (Figure 2a,b). The lactate concentration was different between the groups: The high-intensity (HI) intervention resulted in a higher lactate concentration than both lower-intensity (LI) training protocols

(LI, *p* = 0.003; LI-BFR, *p* = 0.008). In the HI group, the mechanical pain threshold increased from before to after training (*p* < 0.05) (Figure 2c).

**Figure 2.** Objective outcomes of training interventions. Data are displayed as mean and 95% confidence intervals. Bpm, beats per minute; LI-BFR, low-intensity training with blood flow restriction; LI, low-intensity training; HI, high-intensity training. (**a**) Differences in blood lactate concentration pre- and post-training, (**b**) maximal heart rate, and (**c**) differences in mechanical pain threshold preand post-training.

2.3.2. Participant-Reported Outcomes

The perceived exertion was greater during the HI intervention than in the LI interventions (LI, *p* = 0.005; LI-BFR, *p* = 0.028). The HI group scored a lower value than the LI group in the feeling scale (*p* < 0.05). Participants in the LI group reported lower values on the fatigue scale than the LI-BFR group (*p* = 0.028) and the HI group (*p* = 0.004). The corresponding values are displayed in Figure 3.

**Figure 3.** Participant-reported outcomes of training interventions. Data are displayed as mean and 95% confidence intervals. (**a**) Maximal exertion. (**b**) Maximal fatigue. (**c**) Maximal discomfort. NRS, numeric rating scale; LI-BFR, low-intensity training with blood flow restriction; LI, low-intensity training; HI, high-intensity training.

### *2.4. Profiling of Circulating miRNAs*

### 2.4.1. Capillary Blood for miRNA Isolation, Expression Analysis, and Quantification

A single capillary blood draw resulted in ≥50 μL plasma, and, in comparison to venous blood sampling, the average degree of hemolysis differed significantly (*p* < 0.05) as determined by OD414 in a pilot study (Figure 4a,b). Only 50 μL of plasma were sufficient to isolate total RNA and to reverse-transcribe miRNAs for real-time PCR-based quantification. In order to identify stably expressed reference genes for normalization, five candidate miRNAs (hsa-miR-30e-5p, hsa-miR-148b-3p, hsa-miR-222-3p, hsa-miR-425-5p, hsa-miR-484) were tested for stable expression over the entire range of samples being investigated. Except for hsa-miR-222-3p, all miRNAs were suitable for normalization (Figure 4c).

**Figure 4.** (**a**) Example of an OD scan from 200 to 700 nm with a distinct absorbance peak at 414 nm to assess hemolysis. Different lines depict different plasma samples. (**b**) OD414 values in plasma samples after venous or capillary blood draw (\* *p* < 0.05). (**c**) Differences in miRNA abundance of typically detected miRNAs in plasma to determine stable expression pre- and post-training intervention.

#### 2.4.2. Screening of Expression Changes in Circulating miRNAs before and after BFR Training

Based on the assumption that the LI-BFR reduces blood flow to the periphery in a way that is comparable to that of a PAD, eight plasma samples of four participants pre- (control) and post-training were selected and subjected to the human serum/plasma focus panel consisting of 179 miRNA assays targeting human plasma-relevant miRNAs, reference miRNAs, and spike-in controls. A global CT mean of expressed miRNAs was used for normalization, and cel-miR-39-3p was included as an internal amplification control. In each sample, more than 80% of miRNAs surpassed the lower limit of detection of a *C*t < 35 (Figure 5a). Significant miRNA expression changes were visualized in the volcano plot (Figure 5b). A total of 11 miRNAs were selected for further validation due to their markedly altered expression or previous association with collateral growth (Table 1). Interestingly, among the differentially expressed miRNAs identified, three arteriogenesis-associated, previously detected miRNAs were recovered: miR-143-3p, miR-195-5p, and miR-126-5p.

**Figure 5.** qPCR-based serum/plasma focus panel of circulating miRNAs (**a**) Distribution of *C*t values for the processed data of each plasma sample. (**b**) Volcano plot of differentially expressed miRNAs pre- and post-LI-BFR training. Data points outside the two dashed lines are up-regulated (red) or down-regulated (green) more than x-fold. Data points above the solid horizontal line have *p*-values less than 0.05.

**Table 1.** Over-expressed miRNAs (fold regulation values greater than 1.5) and under-expressed miRNAs (fold regulation valuesless than−1.5) detectedin the screen and analyzedin the three different training groups.


\* *p*-value < 0.05, \*\* *p*-value < 0.01, HI: High intensity training, LI-BFR: Low intensity training with blood flow restriction, LI: Low intensity training. Bold text shows the miRNAs that have been previously associated with collateral growth.

### 2.4.3. Analysis of miRNAs in Different Training Intervention Groups

The abundance of these 11 differentially expressed miRNAs was analyzed in each training group in individual assays in a larger cohort of 12 participants. Only miR-143-3p was confirmed to be down-regulated after LI-BFR. In contrast to the initial screening results, miR-139-5p, miR-143-3p, miR-195-5p, miR-197-3p, miR-30a-5p, and miR-10b-5p were up-regulated after HI. There was no differential expression after LI. (Figure 6, Table 1)

**Figure 6.** Differences in miRNA expression pre- and post-training. Data are displayed as mean and 95% confidence intervals. (**a**) LI-BFR, low-intensity training with blood flow restriction. (**b**) HI; high-intensity training. (**c**) LI, low-intensity training.

### *2.5. Associations between Training Outcomes and Circulating miRNAs*

The pre-to-post changes in lactate concentration and miR-143-3p expression showed a significant linear positive correlation (intervention partialized) of *r* = 0.34 (*p* = 0.048). This correlation is visualized in Figure 7. Without considering the group as a partializing co-variate, lactate and miRNA-143-3p differences were associated with a coefficient of *r* = 0.305; however, this correlation lacks statistical significance (*p* = 0.075). No other systematic correlation between lactate concentration and miRNA expressions occurred.

**Figure 7.** Scatterplot diagram of miR-143-3p and lactate concentration with a correlation line (including confidence intervals); LI-BFR, low-intensity training with blood flow restriction; HI, high-intensity training, LI, low-intensity training.
