Fit for comparison: controlling for cardiorespiratory fitness in exercise physiology studies of sex as a biological variable

Abstract

More studies in exercise physiology are investigating sex as a biological variable, but the potential confounding effect of cardiorespiratory fitness is often neglected. As maximal oxygen uptake ( ${\dot{V}}_{O_2\max }$ ) correlates with many physiological outcomes at rest and in response to exercise, differences in ${\dot{V}}_{O_2\max }$ between male and female participants may hinder interpretation. Here we revisit decades-old arguments that advocate for matching males and females for ${\dot{V}}_{O_2\max }$ normalized to fat-free mass (FFM) when investigating sex differences in the context of exercise. The rationale for using FFM to normalize ${\dot{V}}_{O_2\max }$ , as opposed to total body mass, is that females, on average, have a greater proportion of body fat than males and body fat does not contribute to ${\dot{V}}_{O_2}$ . Using a multistudy dataset of males (n = 54) and females (n = 54) matched for ${\dot{V}}_{O_2\max }$ per FFM, we illustrate the different approaches to ${\dot{V}}_{O_2\max }$ normalization and the effects of poor or incorrect matching on interpretation. Modern assessments of body composition allow for segregation of bone from total FFM and regional measures of body composition; however neither approach seems to be an improvement on whole-body FFM as the normalization factor for ${\dot{V}}_{O_2\max }$ . A group-level difference of less than 5% for ${\dot{V}}_{O_2\max }$ per unit FFM is a strong starting point for comparisons between males and females, but the allowable difference depends on the extent to which cardiorespiratory fitness influences the variable of interest and other competing study design decisions. Researchers should be encouraged to normalize ${\dot{V}}_{O_2\max }$ to FFM for exercise physiology studies investigating sex differences.

Keywords: aerobic fitness; body composition; body fat; fat‐free mass; maximal oxygen uptake; research design; scaling; sex differences.

Figure 1. Methods of expressing maximal oxygen uptake (V˙O2max) and their underlying relationships to fat‐free mass (FFM), body mass (BM) and body composition

Part I: ${\dot{V}}_{{\mathrm{O}}_2\max }$ can be expressed in absolute units or relative to BM or FFM to account for differences in body size and composition. Size of icons and circles represent differences in average body mass, and pie charts represent the average difference in body fat percentage for this dataset. Part II: relationships between expressions of ${\dot{V}}_{{\mathrm{O}}_2\max }$ and anthropometric variables. Absolute ${\dot{V}}_{{\mathrm{O}}_2\max }$ (A, D, G) is positively related to FFM and BM and negatively related to body fat percentage. Expressing ${\dot{V}}_{{\mathrm{O}}_2\max }$ relative to BM (B, E, H) accounts for overall size differences between sexes but does not completely remove the relationship to FFM or body fat percentage. Expressing ${\dot{V}}_{{\mathrm{O}}_2\max }$ relative to FFM (C, F, I) removes relationships with both FFM and body fat percentage, accounting for critical sex differences in both size and body composition between sexes. R ² values in black show the overall value (pooled across sexes), whereas values in purple and green show correlations within females and males, respectively. Asterisks (*) indicate p < 0.05. n = 54 males and n = 54 females.

Figure 2. Hypothetical outcomes of sex comparisons using total body mass (BM) or total fat‐free mass (FFM) to normalize and match cardiorespiratory fitness when variables of interest are or are not different between sexes

Part I: sex differences in body composition impact matching males (green) and females (purple) for ${\dot{V}}_{{\mathrm{O}}_2\max }$. Because of the sex difference in the relationship between ${\dot{V}}_{{\mathrm{O}}_2\max }$ per kilogram FFM and ${\dot{V}}_{{\mathrm{O}}_2\max }$ per kilogram BM (A), driven by body composition, matching groups of males and females based on ${\dot{V}}_{{\mathrm{O}}_2\max }$ normalized to BM leads to females having greater ${\dot{V}}_{{\mathrm{O}}_2\max }$ per kilogram FFM (B). This difference caused by ${\dot{V}}_{{\mathrm{O}}_2\max }$ per BM matching is shown as Δ_BM throughout the figure and is responsible for the ‘fitness effect’ shown in panels B, D and F in part II. In contrast, matching groups of males and females based on ${\dot{V}}_{{\mathrm{O}}_2\max }$ normalized to FFM leads to females appearing less aerobically fit, i.e., having lower ${\dot{V}}_{{\mathrm{O}}_2\max }$ per kg BM (C). Part II: the effect of cardiorespiratory fitness matching technique is shown across examples where underlying sex differences do or do not exist. In all examples a strong and influential relationship between the variable of interest and ${\dot{V}}_{{\mathrm{O}}_2\max }$ is assumed (A, C, E). Notably, matching ${\dot{V}}_{{\mathrm{O}}_2\max }$ per kilogram BM across groups leads to false positives (D), false negatives or diminished differences (B) or exaggerated differences (F), depending on the scenario. Matching ${\dot{V}}_{{\mathrm{O}}_2\max }$ per kilogram FFM correctly identifies true sex differences (B, F) or the lack of sex differences (D). In instances where the outcome of interest is weakly related to or independent of cardiorespiratory fitness (not shown here), the effect of poor or unknown matching is likely negligible. In part I n = 54 males and n = 54 females for panels A and C. To match participants by ${\dot{V}}_{{\mathrm{O}}_2\max }$ normalized to BM, some participants were removed for panel B (n = 40 males and n = 40 females). Relationships and means shown in part II are hypothetical, and asterisks (*) indicate when a sex difference would be detected in these hypothetical situations.

Figure 3. The relationship between different expressions of maximal oxygen uptake (V˙O2max) and various body compartment/region masses

${\dot{V}}_{{\mathrm{O}}_2\max }$ is expressed in absolute units (column 1) relative to body mass (BM, column 2) or relative to fat‐free mass (FFM, column 3). Lean mass represents FFM, with bone mineral content (BMC) subtracted. Panels show relationships between ${\dot{V}}_{{\mathrm{O}}_2\max }$ expressions and total lean mass (A–C), leg FFM (D–F), leg lean mass (G–I), leg + trunk FFM (J–L) and leg + trunk lean mass (M–O). n = 54 males and n = 54 females for all panels. R ² values in black show the overall value (pooled across sexes). Asterisks (*) indicate p < 0.05.