*3.1. Subject Characteristics*

Age, height, weight, BMI, and Leg Extension 1 RM for all subjects included in the analyses are provided in Table 3. The control muscle biopsy from one elderly woman was found to show irregularities (one fascicle filled with unusually large and small muscle fibres positive for NCAM, MHCn, and MHCe), and this subject was therefore excluded from all analyses.



*3.2. Tissue Immunohistochemistry at Baseline—Young and Elderly Women*

On average, the numbers of fibres included in the fibre type and size analysis were 212 (129–352) for type I and 151 (68–247) for type II fibres in the young participants. The corresponding values for the elderly were 169 (85–267) type I and 143 (45–487) type II fibres. The type I fibre percentage was 59 ± 11% (35%–74%) for the young and 58 ± 15% (22%–75%) for the elderly, with no difference between them. As seen in Figure 1, the elderly had significantly smaller type II fibres compared to both their own type I fibres (−38%, *p* < 0.001) and the type II fibres in the young (−36%, *p* < 0.001).

On average, the number of fibres included in the immunohistochemical analysis of denervated fibres was 1080 [401–2270]. MHCe-positive fibres were only found in the excluded subject and are therefore not presented. The elderly had significantly more MHCn- and NCAM-positive fibres compared to the young (Figure 2).

No significant differences between the previously exercised and the control leg were found in either the young or the elderly for MHCn or NCAM (online Supplementary Figure S3). We evaluated the fibre size of all transversely cut MHCn- and NCAM-positive fibres from the control leg. A clear majority of the MHCn- and NCAM-positive muscle fibres were smaller than 150 μm2 (online Supplementary Figure S3).

**Figure 1.** Muscle fibre size analysis in biopsy cross-sections from the control legs of 12 young and 11 elderly women. Individual values are displayed and with type I and II values for an individual connected by a dashed line. The type II fibres of the elderly individuals were significantly smaller than their own type I fibres and the type II fibres of the younger individuals. \* *p* < 0.001 vs. young type II, # *p* < 0.001 between fibre types in elderly. Images (**a**–**d**) illustrate the analysis process. (b,d) show representative images of the same area, which has been delineated by the macro in ImageJ. This is an elderly subject with a mean fibre size of 3025 μm2 and 1688 μm2 for type I and II fibres, respectively. Similarly, a and c show representative images of the same area in a young subject with a mean fibre size of 3574 μm2 and 3378 μm2 for type I and II fibres, respectively. MHC1, myosin heavy chain 1. Scale bars = 100 μm.

One biopsy from the exercised leg of a young subject showed 13 NCAM-positive fibres (1.3% of total fibre count) all located adjacent to a thick band of connective tissue, reminiscent of the myotendinous junction (MTJ). Collagen XXII staining confirmed that this was in fact MTJ, so these fibres were not included in the analysis of this biopsy (see online Supplementary Figure S4 for image). One young subject had 13 (1.45% of total fibre count) NCAM-positive fibres, all of which were located at the edge of the biopsy. This area was not stained by collagen XXII and remained NCAM-positive on additional sections and was therefore not excluded from the analysis. In all other samples MHCn- and NCAM-positive fibres were randomly scattered in between normal muscle fibres.

**Figure 2.** Muscle fibres positive for MHCn or NCAM in biopsy cross-sections from 12 young and 11 elderly women. Only the control leg is shown. Individual values are presented with the median (horizontal line). Panels show examples of small MHCn (**a**) and NCAM (**b**), and large MHCn (**c**) and NCAM (**d**) fibres (arrows). Positive fibres are green, dystrophin, red. \* *p* < 0.05 vs. young. MHCn, neonatal myosin heavy chain; NCAM, neural cell adhesion molecule. Scale bars = 100 μm.

### *3.3. Tissue mRNA at Baseline and in Response to Exercise—Young and Elderly Women*

The muscle tissue of the elderly women had significantly lower levels of AChR β1 mRNA compared to the young women, whereas levels of both AChR γ and MHCn mRNA were higher in the elderly compared to the young (Figure 3). Tendencies for differences were seen for gene expression levels of AChR α1 and muscle-specific-kinase (MuSK).

Both the elderly and the young women had a significant upregulation of AChR α1 mRNA in the previously exercised leg compared to the control leg (Figure 3). The exercise response of AChR δ mRNA only reached statistical significance in the elderly. AChR ε mRNA were detected in less than half of the samples at levels very close to detection limit of one molecule and with no preference for any group (data not shown).

**Figure 3.** Gene expression in muscle biopsies of healthy young (*n* = 12) and elderly (*n* = 11) women, at rest (control) and five days after a single bout of one-legged exercise. mRNA data were normalized to RPLP0 and are shown as geometric means ± back-transformed SEM, relative to young control legs (control leg) and own control leg (response to exercise). \* *p* < 0.05 elderly vs. young. # *p* < 0.05 vs. control leg. Tendencies are written. AChR: acetylcholine receptor; MuSK: muscle-specific-kinase; MHCe: embryonic myosin heavy chain; MHCn, neonatal myosin heavy chain; NCAM, neural cell adhesion molecule; GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase; RPLP0: Ribosomal Protein Lateral Stalk Subunit P0.

#### *3.4. Cell Culture at Baseline and in Response to Exercise—Young and Elderly Women*

The fusion index of the cell cultures from the rested and exercised legs of the elderly women was 36.3 ± 4.2% and 36.1 ± 5.0%, respectively, with the corresponding values for the young group being 52.2 ± 1.8% and 49.8 ± 2.2%, respectively (main effect of age, two-way repeated measures ANOVA).

All gene expression targets were more strongly expressed in differentiating compared to proliferating cells (see online Supplementary Figure S5). In the proliferating condition, the cells from the elderly had lower gene expression levels of MHCe and MHCn compared to young (Figure 4). Similarly, we also found a significantly lower level of MHCn gene expression in the differentiating cells in the control leg in the elderly compared to the young. AChR β1, δ, and γ all showed age-related tendencies.

**Figure 4.** Images display cells in the proliferation condition (Desmin, red, and DAPI, blue) and in the differentiation condition (Desmin, red, Myogenin, green, and DAPI, blue), scale bars = 500 μm. Gene expression in cell cultures from the control and exercised legs of healthy young (*n* = 12) and elderly (*n* = 11) women. mRNA data were normalized to RPLP0 and are shown as geometric means ± back-transformed SEM, relative to young control leg (control leg) and own control leg (response to exercise). \* *p* < 0.05 elderly vs. young. # *p* < 0.05 vs. control leg. Tendencies are written. AChR: acetylcholine receptor; MuSK: muscle-specific-kinase; MHCe: embryonic myosin heavy chain; MHCn, neonatal myosin heavy chain; NCAM, neural cell adhesion molecule; GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase; RPLP0: Ribosomal Protein Lateral Stalk Subunit P0.

Differentiating cells from the previously exercised leg from the young subjects demonstrated a lower gene expression of MHCn versus the control leg (Figure 4).

#### *3.5. Tissue mRNA in Response to Exercise—Elderly Men*

In general, gene expression in four out of the five AChR measured demonstrated a response to exercise. AChR α1 mRNA was downregulated 4.5 h and one day after the exercise and returned to baseline in four days (Figure 5). AChR β1 mRNA was downregulated at 1, 4, and 7 days. AChR δ mRNA showed a tendency for a decline 4.5 h after exercise and was upregulated seven days after the exercise. AChR γ mRNA decreased 4.5 h after the exercise bout. No significant exercise-induced changes in gene expression of the AChR ε subunit, MuSK, MHCe, or MHCn were observed.

**Figure 5.** Gene expression in muscle biopsies of 25 healthy elderly men ten days before (baseline) and 4.5 h, one day, four days, and seven days after a single bout of exercise. mRNA data were normalized to RPLP0 and are shown as geometric means ± back-transformed SEM, relative to baseline. \* *p* < 0.05 vs. baseline. Tendencies are written. AChR: acetylcholine receptor; MuSK: muscle-specific-kinase; MHCe: embryonic myosin heavy chain; MHCn, neonatal myosin heavy chain; NCAM, neural cell adhesion molecule; GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase; RPLP0: Ribosomal Protein Lateral Stalk Subunit P0.
