The current study shows that there are sex differences in outcome measures for these commonly used assays. Specifically, we show voluntary wheel running, females ran greater distance and total time than males, consistent with prior studies [22, 43, 44]. Uniquely, we show that males and females initiate running bouts an equal number of times, but both average and max bout duration is greater in females, which likely contributes to greater distance and time run observed in females. In forced treadmill running, females ran for a significantly longer period, but obtained the same top speed as males. In open field testing there were no sex differences in total distance and speed, but there were sex differences in active time. Thus, each assay appears be a unique measure of activity with voluntary wheel running showing the largest sex-specific differences. Importantly, if these assays are being utilized to test new drugs or animal models researchers should consider accounting for sex differences as they could potentially mask the true findings in an experiment.
Voluntary Wheel Running
One of the strengths of voluntary wheel running is that it is a measure of self-initiated activity in a non-stressful environment [25, 45–47]. Activity is recorded during the dark phase without any human interaction and occurs during their naturally active period. The use of computer-based data collection allows for a more complete analysis of voluntary wheel running behavior and may correlate to physical activity levels in human subjects [48]. However, because wheel running is self-selected, there is no control or standardization of the task, and thus total activity varies between each individual animal.
Consideration of sex differences in wheel running behavior began in the early 1920's and numerous data support prominent sex differences in rodents; specifically, females run a greater distance and total time than males [43, 44, 49]. We expanded these results by showing a similar number of bouts between sexes, with females showing greater bout duration, greater speed, and greater rate of increase to plateau, all of which contribute to greater distance run.
A bout is defined as a brief period of increased activity and has been considered an important component of physical activity [1, 4]. For example, the American College of Sports Medicine recommends individuals accumulate 150 minutes of moderate to vigorous activity per week with bout durations of at least 10 minutes. However, clinicians often recommend increasing physical activity levels regardless of bout duration [50, 51] and prior research suggests improvements in function and pain, regardless of bout duration [52, 53]. The current study showed a similar number of bouts per day between sexes, but longer bout duration in females. Data were captured in 1-minute intervals, and bouts were separated by at least one minute without running wheel activity. Conversely, De Bono et al. showed females ran a greater total number of bouts but showed no sex differences in bout duration – data were collected in 5-second intervals, but it is unclear precisely how bouts were determined. This difference is likely related to how bouts were collected and calculated and could represent the difference between a technical bout and a biologically meaningful bout of activity. Prior work in humans have shown that bouts of < 10 minutes are associated with reduced frailty in both sexes [52], but activity intensity is a greater determinant of cardiometabolic risk than bout duration [54]. Total activity time regardless of bouts is related to fatigue, function, and disease severity [37], while the total minutes spent in ≥ 10-min bouts is associated with lower pain [55] in individuals with fibromyalgia, suggesting both total activity and bouts are important in clinical populations. Future work in preclinical studies is needed to determine meaningful bout-lengths.
The current study considered two phases of running wheel behavior; an acclimation phase during which animals increase their running distance each day, and a plateau phase where daily running distance has normalized. The current study showed that both male and female mice reached a plateau after 2 weeks for daily distance but varied for other measures. Total running time normalized after 5 days, total bouts after 3 days, and average and peak bout duration between 8–14 days. These data are consistent with prior studies who showed that speed of wheel running peaked within the 2 or 3 weeks in both sexes [26, 28], but contrast others showing shorter durations for acclimation and more prominent sex differences [25, 27]. Differences could be related to the type of running wheel used, the external environment, or age of the animals.
Longer-term acclimation is frequently performed to normalize wheel running. However, this is potentially problematic given the impact of voluntary wheel running, as a form of exercise, on physiological responses in multiple systems including body composition, muscular system, metabolic capacity, peripheral and central nervous system, and immune system [8, 22, 46, 56–61]. Physiological changes begin immediately with exercise and can produce biological effects within days [9, 46, 60, 62]. For example, running wheel activity in mice can prevent the development of chronic muscle pain and associated changes in the central nervous system with just 5 days of activity [60]. Therefore, if experimental mice undergo 2–3 weeks of wheel running to normalize running distance, it is likely these mice undergo significant physiological adaptations that alter the responses observed in otherwise sedentary mice.
Forced Treadmill Running
Forced treadmill running is utilized to determine maximal exercise capacity and allows for control of speed and intensity. To maintain running, electric shock is often used to maintain running and can cause stress [21, 24]. The current study showed females ran 1.5 minutes, 4.3%, longer than males. This difference was statistically significant but whether the difference is biologically meaningful is debatable. As a secondary analysis we included the animals that were available, but for perspective, at an effect size of 0.46 and an alpha of 0.05 we needed an n = 75 to achieve 0.8 power. In contrast to our data, prior studies report female mice run 25–50% longer than males [31, 32] but female rats run 40% less than males [33]. These differences are likely related to differences in the protocols, species (rat vs. mice), different treadmill inclines (10 vs, 20-degrees), time spent at lower speeds, or rate of speed increase.
Open Field Testing
The current study showed no differences in activity between sexes for open field testing parameters of distance and speed, but there were significant differences in active time. These data generally agree with prior studies in C57BL/6J mice who show equivalent activity between sexes [29, 35, 36]. On the other hand, other mouse strains, MOLF and SJL, show higher activity in females [34]. Complicating comparison between studies is differences in individual testing parameters, including size of the open field chamber, lighting, and the transparency of the walls (clear versus opaque). While activity assessment of the 30-minute data in aggregate yielded no meaningful differences in distance and speed, a more in-depth analysis examining data in 5-minute segments revealed unique-sex-specific differences. During the final 20 minutes of the test females maintained running distance and speed similar to the first 10 minutes, while males showed steady decline during the last 20 minutes. This is a small but noteworthy effect, as it parallels the pattern of decreased duration of running in males that we see in voluntary wheel running.
Mechanisms of sex differences in activity assays
Sexually dimorphic differences in activity might be due to variation in sex hormones, muscle capacity, or age. Multiple studies show increases in estradiol enhance wheel running activity in male and female mice [32, 63–67], but not exploratory behavior in the open field test [36, 68, 69]. Further, the sex-differences in total distance, total duration and speed of wheel running behavior disappears by 6–9 months of age [47]. Female mice show higher mobilization and use of lipids within the skeletal muscle than males and have more type I and less type II muscle fibers, which together could contribute to greater endurance [31, 32]. Thus, the sex-specific differences likely involve multiple mechanisms across systems.
Strengths and Limitations
This study was a secondary analysis of baseline data, and thus was not designed specifically to test sex differences. However, protocols were consistent across animals in each cohort and data was taken at baseline prior to any intervention. Separate mice were utilized for each assay; therefore, we were unable to correlate relationships within-mouse between the different activity assays. Similarly, we did not account for muscle volume or cross-sectional area. We collected wheel revolutions every minute. While common practice, extrapolating speed and bout data is less granular than previously reported by de Bono who collected every 5 seconds [25], and thus may be difficult to directly compare results between studies.