## 4 Discussion  
The goal of this study was to identify possible differences between common approaches based on the CW-NIRS signal slope to estimate the muscle oxygen consumption, which is of special interest when, e.g., discussing the outcome of a study in the context of existing literature. Five slope-based approaches were analyzed that either use a single signal (HHb, O\({}_{2}\)Hb) or a combination of different signals provided by the CW-NIRS device (TSI, HBdiff, HBmean). This study showed significant differences between three out of the five analyzed approaches to estimate muscle oxygen consumption of the same contraction data. More precisely, differences were found between all approaches except for TSI vs. HBmean as well as HBdiff vs. HHb. However, O\({}_{2}\)Hb always differed from the other approaches resulting in an underestimation of mVO\({}_{2}\) values and additionally showed the biggest percentage difference (Table 1) compared to the rest. The difference between O\({}_{2}\)Hb and HHb is particularly unexpected. This study used a pressure cuff to exclude blood volume changes in the region of interest. In such a closed system, the O\({}_{2}\)Hb and HHb signals are assumed to be mirrored and, hence, should have the same absolute slope [11]. Having alterations of blood volume can influence the estimated mVO\({}_{2}\). In literature, this has been described as an increase in tHb [21; 28; 35]. However, the analysis showed that this was not the case and, instead of an increase in blood volume, there was a decrease in blood volume (Figure 3). Similar trends are shown in [PERSON] et al. (Figure 4 in [28]). With a maximum difference of 1.8 uM in the current analysis, the observed differences are quite small and, therefore, \begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Bias [\%]** & **LoA [\%]** & **Range [\%]** \\ \hline TSI and HBmean—Hbdiff and HHb & \(-1.9\) & \(11/-14.8\) & \(12.9\) \\ \hline TSI and HBmean—O\({}_{2}\)Hb & \(-1.4\) & \(5.2/-7.9\) & \(6.6\) \\ \hline HBdiff and HHb—O\({}_{2}\)Hb & 0.3 & \(11.0/-10.4\) & \(20.4\) \\ \hline \end{tabular}
\end{table} 
Table 2: Results of the Bland–Altman plots comparing the different methods. Figure 5: Bland–Altman plots. Left plot: comparison of TSI + HBmean and HBdiff + HHb. Middle plot: comparison of TSI + HBmean and O\({}_{2}\)Hb. Right plot: comparison of HBdiff + HB and O\({}_{2}\)Hb. Note: dashed horizontal line represents the bias [%] and dotted horizontal line represents the limits of agreement (\(\pm 1.96\) SD) of the respective comparison. For parameters with a plus sign (e.g., TSI + HBmean), the mean value was calculated because statistics showed no significant differences between those parameters. can be assumed to be negligible, though they are statistically conspicuous. Consequently, these differences in tHb cannot explain the differences observed in mVO\({}_{2}\). Despite significant absolute differences, the question concerning the relative agreement of the different approaches remains. The bias between the comparisons reached a maximum of \(\sim\)2%, revealing a good accuracy between the different approaches. Besides this, the limits of agreement showed a broader range across the comparisons, identifying the least precision for HBdiff and HHD vs. O\({}_{2}\)Hb. For this comparison, 95% of the observations were within a range of \(\pm\)20.4%. On the other hand, TSI and HBmean vs. O\({}_{2}\)Hb showed a small bias accompanying the highest precision. Hence, O\({}_{2}\)Hb underestimated the values; however, 95% of the observations were within a range of \(\pm\)6.6%. A limitation of this study is that we only used one submaximal intensity to estimate oxygen consumption. Therefore, it remains unclear if these observed differences regarding the estimated oxygen consumption are the same across different intensity levels. In the literature there are several papers published investigating the amount of oxygen consumption across different intensity levels using different muscle groups and incremental steps of 10% [11; 21; 36; 37].