Journal of Experimental Biology

Because hypoxia and low temperature independently alter metabolism and reproductive investment, their interaction provides a tractable framework for testing whether combined stressors produce non-additive physiological and reproductive effects. Here, we investigated the single and combined effects of hypoxia and low temperature in Drosophila melanogaster across multiple genetic backgrounds. We quantified metabolic rate, thermal tolerance, body mass, fertility, oogenesis progression, and oocyte apoptosis to assess organismal responses to environmental stress. Hypoxia generally increased respiratory quotient and body mass, but its effects on thermal tolerance and fertility were highly genotype dependent. Across traits, combined stressors frequently produced responses that differed from those observed under single stressors, including reduced fertility, altered oogenesis, and changes in oocyte cell death. Importantly, these effects were not uniform: some genotypes exhibited increased oocyte production or reduced cell death under combined stress, highlighting pronounced genotype-dependent differences in stress sensitivity and reproductive allocation. Together, our results demonstrate that the interaction between hypoxia and temperature can modulate metabolic and reproductive responses in ways that are not predictable from single-stressor responses alone. These findings highlight the importance of incorporating genetic background and interacting environmental stressors when evaluating organismal tolerance and adaptive potential under ongoing environmental change.

Variation in mitochondrial aerobic metabolism has been suggested to underlie variation in interindividual performance. Mitochondrial efficiency quantifies, directly or indirectly, the amount of adenosine triphosphate (ATP) produced relative to O2 consumption. High mitochondrial efficiency is theoretically beneficial by providing more ATP per amount of resource consumed, but may come at the cost of increased reactive oxygen species (ROS) production damaging tissues through oxidative stress. Mitochondrial efficiency is a plastic trait but how it changes through postnatal development remains unknown. We hypothesized that strong selective pressure could lead to an increased mitochondrial efficiency to support fast growth but incur an oxidative cost. We tested this hypothesis by quantifying mitochondrial aerobic metabolism, efficiency and ROS production through postnatal growth in Japanese quail (Coturnix japonica), in two highly aerobic tissues: skeletal and cardiac muscles. Mitochondrial efficiency was indeed higher during peak growth in both tissues, but this was surprisingly associated with markedly lower ROS production. This high efficiency was likely achieved via both a lower proton leak and a higher contribution of complex I to respiration. These results show that enhancing mitochondrial efficiency may be important to support growth, but suggest the presence of unexpected ROS mitigation processes during early-life growth.

Extreme climatic events impose considerable stress on organisms with consequences for key ecological interactions such as pollination. Because temperature directly affects metabolic processes, heat variations may also importantly influence the nutritional needs and feeding choices of animals. Here, we studied the effects of thermal stress on the nutritional choices and performances of bumblebees, using a 3D nutritional geometry design. At optimal temperature for colony development (30{degrees}C), bees successfully balanced carbohydrate, protein, and lipid collection, at levels beneficial for body weight and survival. Under cold stress (20{degrees}C), bees reduced their overall nutrient collection while selecting proportionally more carbohydrates, thereby prioritizing survival over weight gain. Under heat stress (35{degrees}C), nutrient balancing was disrupted and survival dropped. Notably however, across all temperatures, bees maintained stable lipid collection while flexibly adjusting the amount of carbohydrates and proteins, suggesting strong constraints on lipid regulation. Given the pivotal role of bees for pollination, identifying how their nutritional needs change in response to climatic conditions is of prior importance for food safety and the conservation of terrestrial ecosystems.

Many marine invertebrates are characterized by broad and highly plastic thermal limits, though the dynamic molecular mechanisms that enable extended thermal acclimation remain poorly understood. A classic example is the green crab (Carcinus maenas), which is a prolific and damaging non-indigenous species. Using a 22-day thermal exposure to cold (5{degrees}C), ambient (13{degrees}C), or warm (30{degrees}C) temperatures, we characterized plastic shifts in C. maenas performance using respirometry and time-to-right. We then used untargeted metabolomics and lipidomics analysis of heart tissues from days 4 and 22 to identify the molecular mechanisms underpinning plastic responses over time. Crabs at 30{degrees}C exhibited higher oxygen consumption rates than counterparts at 5{degrees}C. Interestingly, oxygen consumption rate increased over time at both temperatures, indicating thermal plasticity of aerobic respiration. Temperature-dependent metabolic reprogramming was employed by crabs to sustain aerobic respiration across temperature. Catabolism of branched-chain amino acids was important for energy production at elevated temperatures, while catabolism of arginine may have sustained the minimal energy needs of crabs exhibiting metabolic depression at cold temperatures. Righting response was positively correlated with temperature, and did not exhibit any changes over time. Lipidome remodeling consistent with homeoviscous adaptation could have enabled motor activity across temperature. Higher abundances of saturated and monounsaturated lipids likely provided structural integrity to cell membranes at 30{degrees}C, while lower abundances of these compounds may have enabled membrane fluidity at 5{degrees}C. Our work demonstrates the importance of ongoing molecular reprogramming in long-term acclimation, even when whole-animal physiology remains relatively stable. Summary StatementThis study demonstrates how the highly invasive green crab regulates metabolite and lipid pathways over time to maintain physiological performance across different temperatures.

Arboreal insects have developed various strategies to navigate their discontinuous habitats. Many insects, including leafhoppers, katydids, and praying mantises, exhibit the ability to actively leap across their leafy platforms and land on a distant substrate. This behavior is especially important for non-winged insects, including nymphal forms of winged insects, which cannot fly between these substrates. To make a targeted jump, an animal must first orient towards the target, estimate the target distance and angular location, and jump with the appropriate take-off speeds and angles to land on their intended substrate. In three-dimensional space, jumping from one point to another requires estimating distance, as well as azimuthal and elevational angles. Jumping insects such as mantises typically reorient their bodies on the substrate to align with the azimuthal direction of the target. This behavior effectively reduces the task to a two-dimensional problem, in which they must estimate only the distance to the target and its elevational angle. Many insects, including praying mantises, perform rhythmic lateral head movements called peering before performing a targeted jump. Although previous studies suggest that mechanisms such as motion parallax while peering are used for distance estimation, the full repertoire of behaviors that enable mantises to jump to arbitrarily located substrates remains unclear. We hypothesized that mantises have distinct behaviors for distance and elevation angle estimation, which enable them to independently modulate their take-off speeds and angles before jumping. To test this hypothesis, we developed behavioral assays in which mantises were placed on a launch platform and jumped to a target platform positioned at variable distances and angles. Using this apparatus, we filmed the jumps of Giant Asian mantis nymphs (Hierodula spp.) with high-speed videography and tracked body parts to quantify take-off speed and angle. Because mantis jumps are ballistic, their trajectories can be modeled as projectile motion. Our results indicate that mantises estimate target distance and elevation angle using two separate behavioral strategies: distance is assessed through peering maneuvers that generate motion parallax, whereas elevation angle is determined through visual fixation of the target accompanied by specific postural adjustments. By combining these behaviors, mantises modulate the magnitude and direction of propulsive force to achieve successful jumps.

Although predation is a major driver of group living across taxa and the antipredator benefits of grouping are well established, the energetic costs experienced by groups under predation remain largely unexplored. In the current study, we use wild, white mullet (Mugil curema, Valenciennes 1836), to provide the first real-time quantification of the energetic cost of escape in schooling fish using intermittent, closed-loop respirometry. We found that small groups exposed to predators showed a 53.8% increase in their organismal metabolic rate (MO2) as compared to groups without predator exposure. When we evaluated antipredator behaviors such as escape response, group cohesion, and displacement of the group centroid, we found a positive correlation to energetic costs. We then investigated whether escape responses are socially modulated by comparing the energetic costs of escape across solitary individuals, solitary individuals with visual access to a group, and groups. We found that escape frequency and energetic costs to predation were comparable across social contexts, indicating that escape may be an intrinsic survival response independent of cues from group members. Furthermore, we found that fish exposed to predators showed markedly reduced feeding, suggesting that predation constrains energy acquisition in addition to imposing direct energetic costs. Our results provide the first direct quantification of the energetic costs of escape in a schooling fish, offering new insights into the physiological trade-offs underlying collective antipredator defenses.

Winter survival in dormant animals depends on conserving finite energy reserves, yet winter temperatures fluctuate around shifting means. In ectotherms, metabolic rate increases exponentially with temperature, so thermal variability is expected to accelerate energy loss, with important consequences for overwinter survival and population persistence under climate change. However, it remains unclear whether dormant ectotherms can compensate physiologically for thermal variability. We overwintered Bombus impatiens queens under constant (2, 3, 4{degrees}C) or variable (2 {+/-} 6{degrees}C or 4 {+/-} 6{degrees}C) regimes for six weeks, then measured metabolic rates across a range of temperatures. The temperature dependence of metabolic rate shifted in response to thermal experience, but the direction of compensation depended on mean temperature: variability centered on 2{degrees}C elevated metabolic rate and increased thermal sensitivity relative to all other conditions, whereas variability centered on 4{degrees}C reduced metabolic rate and dampened thermal sensitivity relative to constant 4{degrees}C. We used these metabolic responses to simulate rates of lipid depletion and found that survival trajectories echoed physiological shifts: experiencing variability around 2{degrees}C would reduce subsequent survival time, whereas experiencing variability around 4{degrees}C would preserve subsequent survival even under variable future conditions. Thus, identical thermal variance produced opposite energetic outcomes depending on the mean temperature around which fluctuations occurred. Integrating both temperature means and variability is, therefore, essential for predicting overwintering survival in a changing world.

Multimodal communication plays a central role in animal behavior, particularly when individuals must integrate information from different sensory channels to make rapid decisions. In aquatic environments, chemical and visual cues differ markedly in their spatial and temporal properties, such that chemical signals may be constrained by limited spatial resolution and temporal instability, potentially requiring visual information to reliably guide social decisions. In decapod crustaceans, both cue types are known to mediate reproduction, yet their relative contribution to mate-location behavior remains unclear. Here, we tested how visual and chemical cues from males influence mate-location behavior in females of the prawn Macrobrachium rosenbergii. Females were placed in a central arena and exposed to four stimulus configurations combining visual cues (a life-size photograph of a male or a control background) and chemical cues (water from an aquarium with or without a male). Attraction was quantified as the time spent in each half of the arena. Females showed no directional preference when exposed to chemical cues alone or when visual and chemical cues were spatially incongruent. In contrast, females spent significantly more time near male-associated stimuli only when visual and chemical cues were spatially congruent. These results indicate that mate-location behavior in this species depends on multimodal integration with a strong contextual dependence on visual information, which appears to gate the effectiveness of chemical cues. Spatially congruent multimodal signals are therefore necessary to guide orientation during mate search, suggesting that disruption of visual or chemical information in aquaculture systems may impair mating efficiency.

O_LIEctotherms in thermally variable environments mediate energy expenditure through both physiological and behavioural responses. However, many studies focus on constant temperature acclimation, and few consider behaviour and physiology in unison. It is unclear how acclimation to thermal variability affects locomotory choices, activity timing, and performance across daily thermal cycles. C_LIO_LIWe investigated the effects of thermal variability in the temperate dung beetle Onthophagus taurus. Following acclimation to a low amplitude (22{degrees}C {+/-} 2{degrees}C) or a high amplitude (22{degrees}C {+/-} 10{degrees}C) temperature regime, we measured behaviour and metabolic rate across temperatures. We hypothesised that O. taurus adjusts its locomotive strategy and search window when kept in high amplitude fluctuating temperatures to reduce energy loss associated with high temperature exposure. C_LIO_LIWe found that differences in energy expenditure were determined by propensity for flight which differed between acclimation treatments, particularly at intermediate temperatures. We also found that, following acclimation to a high amplitude of thermal variability, O. taurus exhibited a greater intensity of activity over a narrower window of time, and O. taurus acclimated to a low amplitude of thermal variability showed nocturnal activity. C_LIO_LIWe then used the data to model activity through the growing season over five years. Biophysical models were built using NicheMapR Microclimate and Ectotherm functions to test the length of potential searching time across seasons, the temperatures individuals are exposed, and locomotive strategy. Model outputs showed that acclimation to higher amplitudes of thermal variability increased accumulated degree-hours of activity relative to the low variability acclimation group. Individuals acclimated to higher amplitudes of thermal variability showed greater accumulated degree-hours in spring and fall, but exhibited shorter periods of activity during summer, with the model predicting increased opportunities for flight. Comparatively, O. taurus from the low variability acclimation treatment showed increased night activity in summer but did not fly. C_LI

Linking animal movements to environmental drivers is essential for understanding ecological processes and anticipating species responses to climate change. We investigated habitat-specific movements in a globally significant aggregation of loggerhead turtles (Caretta caretta) nesting in Cabo Verde. Satellite tags on 15 adults (12 females, 3 males) provided multi-year tracks spanning breeding, migration, and foraging habitats. Movements and phenology differed by habitat. During the breeding season, females used either coastal areas, remaining within [~]20 m depth, or undertook long looping forays up to 360 km. Males showed two strategies: two remained resident in Cabo Verde waters, including Fra, the largest male tracked (Curved carapace length of 105 cm compared with a male mean of 90.7 {+/-} 10.3 cm), while the third migrated annually to distant foraging grounds and returned ahead of the subsequent breeding season. In foraging habitats, turtles adopted neritic or oceanic strategies: neritic turtles remained localised in warm, productive waters, whereas oceanic turtles ranged widely in deeper, less productive areas. Time- and space-shift analyses showed that oceanic foragers used intermediate sea surface temperature and chlorophyll-a conditions relative to nearby or temporally shifted alternatives, consistent with movement within a thermal-trophic trade-off. Together, these results show how sex, body size, and energy balance drive habitat-specific movement dynamics in a changing ocean.

Muscle mass significantly influences skeletal muscle behaviour, potentially explaining why traditional massless Hill-type models struggle to predict the forces generated by larger muscles during dynamic, submaximal contractions. However, the applicability of mass-enhanced Hill-type models in human locomotion remains unexplored. Here, we compared the predicted force from a 1D mass-enhanced Hill-type muscle model with a traditional 1D massless Hill-type muscle model across a range of experimentally measured human movements. Kinematic and electromyographic data were collected from twenty participants performing locomotor tasks and supplemented with existing cycling data. Muscle size was geometrically scaled by factors from 0.1 to 10, which causes lengths to be scaled proportionally, cross-sectional area and peak isometric force F0 with the square, and mass with the cube of the factor. Muscle tissue mass (inertia) and cadence increased the differences between mass-enhanced and massless predictions of force and power. At high cadence and the largest scale, the normalized root mean square difference between force traces reached 7% of F0, (averaged across muscles). However, differences between models were minimal (<1%) at human-sized scale 1. Real muscle additionally deforms in 3D, we still do not know the extent to which this extra dimensionality affects muscle forces for these human movements.

Escape responses are a critical behavioural mechanism influencing survival during predation events. In most species of teleosts and several other lower vertebrates, these responses are triggered by Mauthner cells (M-cells), which generate faster escapes (characterised by higher turning rates and shorter response latencies) than non-M-cell triggered responses. Most adult elasmobranchs lack M-cells and consequently exhibit slower escape response timing than teleosts. Spotted Ratfish (Hydrolagus colliei) are a notable exception in that adults possess M-cells, yet their escape response performance has not been explored. Here, we quantify the kinematics and timing of ratfish escape responses elicited by a mechano-acoustic stimulus. We show that ratfish exhibit higher turning rates and shorter response latencies than other adult chondrichthyans, though their response latencies are also significantly longer than those of teleosts. These findings suggest that retention of M-cells confers enhanced escape performance in ratfish, with important implications for their vulnerability to predator attacks. Summary statementThis study reveals that adult Spotted Ratfish (Hydrolagus colliei) show fast escape response with a performance that is intermediate between teleosts and previously studied elasmobranchs.

Aerodynamic performance in airplanes and flying animals can be controlled by changes in wing shape and size, but during flapping flight another key component is wing motion. Observations of free-flying animals reveal natural wing motions, but testing causal mechanisms requires controlled manipulation of flapping kinematics. In flapping wings, aerodynamic interactions between the two wings are expected to depend on wingbeat phase, wing proximity, and wing attitude. How these interactions influence force production remains unclear. Here we used a robotic flapping wing and quantitative flow measurements to test the effect of wing interactions between wings flapped above or below the body in combination with pitch timing during upstroke-downstroke transitions. We show that both force magnitude and direction depend strongly on these parameters. High wing position combined with early pitching enhanced vertical force, whereas low position and late pitching increased thrust. Maximum aerodynamic efficiency was achieved flapping around the horizontal plane and pitching late. Transition phases strongly affected thrust generation and produced substantial body pitch torques. These findings demonstrate that small kinematic adjustments can markedly alter aerodynamic performance and be used for tailless flight control. This offers mechanistic explanations for observed animal wing motions and novel strategies for controlling flapping drones.

Judgment bias tasks are increasingly used to assess affective states in animals, yet the extent to which they might reflect transient states or stable traits remains unclear. Here, we tested bumblebees (Bombus terrestris) in an active choice task across three repeated sessions to assess individual consistency in the absence of any manipulation. Bees were trained to associate each of two colours with either a high or a low reward, presented in separate chambers. During testing, they were presented with ambiguous colours. Bees were more likely to choose the high reward chamber and to choose more quickly in response to colours closer to the positive colour. The latency to choose the cues showed significant and moderate repeatability across sessions, suggesting a stable, trait-like underlying component. In contrast, the repeatability of the chamber choices was negligible, indicating that such responses might be largely state-dependent and influenced by situational factors. These findings suggest that judgment biases, particularly as assessed through an active choice task reflect states affected by external factors. Active choice tasks may help disentangle stable behavioural traits from transient affective states in invertebrates.

Capturing and handling wild animals is essential for ecological and evolutionary research, yet their effects on physiology, behaviour, and reproductive success remain poorly understood. We investigated short- and longer-term consequences of a capture-handling-restraint protocol in wild great tits (Parus major) over three breeding seasons. To assess short-term responses, we measured circulating corticosterone, a metabolic hormone that responds to unpredictable challenges, and automatically recorded provisioning behaviour. We also explored whether environmental and individual traits were related to provisioning latency (i.e., time to resume provisioning after capture). To evaluate longer-term effects, we monitored provisioning in the days following capture and related it to reproductive success (fledgling number and body condition). We predicted that longer handling would increase stress-induced corticosterone and provisioning latency, that these variables would be positively correlated, and that higher corticosterone and longer latencies would be associated with lower reproductive success. After capture, great tits showed elevated corticosterone and delayed provisioning. Contrary to our predictions, handling duration was negatively associated with stress-induced corticosterone in males (but not females) and did not affect provisioning latency. Provisioning latency was unrelated to corticosterone, environmental, or individual variables. Following capture, parents resumed provisioning, and short-term responses had little influence on reproductive success. We show that parental behaviour and physiology are affected by capture restraint protocols on the short term, but offspring condition and survival are not. However, these results should be interpreted cautiously, as our study lacks an uncaptured control group. Our findings highlight that evaluating welfare impacts requires rigorous study design incorporating both immediate and longer-term behavioural and fitness effects.

Understanding how fish navigate complex natural environments requires bridging fine-scale biomechanics with ecological behavior. We investigated the volitional movement and energetics of wild red drum (Sciaenops ocellatus) across laboratory, mesocosm, and field settings. Using flow-respirometry, we quantified metabolic costs and swimming kinematics under ecologically relevant flow conditions shaped by bluff bodies mimicking mangrove roots and oyster mounds. Fish swimming in turbulent wakes exhibited reduced oxygen consumption and altered tailbeat dynamics, especially at high flow speeds. In a large outdoor mesocosm, dual accelerometers revealed a rich behavioral repertoire, including maneuvering and rest, which is not easily observable in confined lab settings. Spectral analysis and clustering identified eight distinct locomotory states, highlighting the limitations of summed acceleration metrics. Field telemetry tracked wild red drum across a 54 km estuarine corridor for a three-year period through an array of 36 acoustic receivers, revealing movement patterns shaped by tidal flow and physical habitats. Hydrodynamic modeling revealed that while laboratory trials demonstrated substantial energetic savings at high flows (approaching 100 cm/s), wild fish were detected predominantly in low-velocity microhabitats (<30 cm/s) near structurally complex features. This mismatch suggests that habitat selection is an adaptive strategy driven by ecological factors such as foraging opportunities, predation refuge, and site fidelity, rather than hydrodynamic efficiency alone. Our multi-scalar approach demonstrates that while flow-structure interactions can reduce locomotor costs for fish, habitat use in the wild reflects broader ecological constraints, offering a framework for integrating biomechanics, physiology, and ecology in conservation-relevant contexts.

Factors varying along elevational gradients impose strong aerodynamic and physiological constraints on powered flight, yet the internal anatomical correlates of flight performance in animals under such conditions remain poorly understood. In hummingbirds, sustained hovering requires extreme muscular power output, making the pectoralis muscle a key interface between environmental constraint and performance. We tested whether elevation is associated with variation in pectoralis microanatomy across three hummingbird assemblages spanning a [~]1500 m gradient in the Colombian Andes. Using tissue morphometry of trichrome-stained transverse sections of the pectoralis, we measured interstitial collagen fraction as a proxy for extracellular matrix investment and quantified fiber cross-sectional area, packing density, and size heterogeneity. Collagen investment varied across elevational bands, peaking at mid elevation ([~]1750 m) and declining toward high elevation ([~]2600 m). In contrast, muscle fibers were smaller and more densely packed at higher elevations. Variation among species was small relative to differences among elevational assemblages. Formal model comparisons provided limited support for non-linear responses to elevation, indicating that patterns across traits are better explained by interacting constraints than by a single monotonic response to factors varying along elevational gradients. These results show that hummingbird flight muscle microanatomy varies with elevation in a trait-specific manner, with the strongest evidence in fiber geometry. More broadly, our findings highlight that multiple components of muscle microarchitecture, including the extracellular matrix, vary in a context-dependent manner across elevational gradients in an extreme volant system.

Animals must continually balance foraging with the risk of predation. In complex natural environments, this means quickly distinguishing between threats and harmless situations. We investigated how site-associated coral reef fishes decide to escape in response to visual cues mimicking predator attacks, using controlled underwater presentations of looming stimuli at varying speeds. We measured escape responses across species and social contexts, comparing them to predator attack speeds observed in the same habitat. Escape responses were highly sensitive to the speed of the looming stimulus, with no responses occurring at low speeds. The speeds triggering escape matched those of predator attacks, whereas cruising swim speeds never triggered a response. Species employed distinct antipredator strategies: Brown Chromis foraged away from shelter with high responsiveness, whereas Bicolor Damselfish remained shelter-dependent with lower escape propensities. Contrary to expectations, the social factors did not affect responses in this study. These findings demonstrate that reef fish are highly sensitive to the approach speed of objects, with species-specific strategies further shaping behaviors. By combining realistic visual threats with natural predator attack data, this study offers insight into how animals make escape decisions in complex, real-world environments.

Iron is an essential nutrient for all types of organisms, including insects and the microbes that infect them. We predicted that insects fed an iron-supplemented diet would accumulate more iron in their hemolymph, and, because infectious microbes acquire iron from their hosts, that this extra iron would increase the severity of bacterial infections. To test this hypothesis, we studied the effects of dietary iron supplementation on infection outcomes in Manduca sexta (tobacco hornworm). Larvae were fed an artificial diet, with or without antibiotics, or the same diets supplemented with 10 mM iron. Control and iron-treated larvae were inoculated with non-pathogenic Escherichia coli or the entomopathogenic Enterococcus faecalis, and bacterial load and larval survival were measured. We found that dietary iron supplementation increased the iron content of hemolymph by approximately 20 fold; however, contrary to our prediction, this increase in iron did not result in an increase in the bacterial load of either E. coli or E. faecalis. The effect of iron supplementation on survival was more complicated. As expected, for larvae inoculated with nonpathogenic E. coli, iron supplementation had no effect. For larvae inoculated with E. faecalis, the effect of iron supplementation depended on whether antibiotics were present in the diet. Without antibiotics, iron supplementation prolonged larval survival; with antibiotics, iron supplementation decreased larval survival. The results of this study do not support the hypothesis that dietary iron supplementation increases infection severity in M. sexta. Instead, the results support the viewpoint that the relationship between dietary iron and infection outcome is complex.

Mass is a fundamental aspect of muscle contractile function, yet the inertial effects of inactive muscle mass is generally neglected in modeling and not quantified in studies on small muscles or isolated fibers. However, during submaximal contractions, inactive muscle tissue may take longer to be accelerated by active fibers, and may be subject to prolonged deceleration, both of which may potentially reduce force development and work output. We sought to test if inactive tissue mass imposes an inertial penalty on muscle performance, using in situ sinusoidal work-loop experiments on rat plantaris muscles. Regional fascicle dynamics, measured across supramaximal and submaximal levels of activation, showed that decreasing activation significantly reduced fascicle strain and increased both shortening and lengthening latency. Contrary to our predictions, however, reductions in work, beyond those explained by decreased fascicle strain, were negligible. Normalized work did not decline disproportionately relative to force, suggesting no clear inertial penalty on work at this muscle size. Our findings suggest that while inactive muscle mass influences the dynamics of submaximal contractions, its impact on work during submaximal contractions at small muscle sizes is limited.