In the Netherlands, in the current forensic medical practice, estimation of the post-mortem interval (PMI) is mainly based on the state of body cooling (algor mortis), post-mortem body stiffness (rigor mortis) and post-mortem lividity (livor mortis) [1,2]. A less used method in the Netherlands is based on supravital muscle reaction (SMR) by mechanical stimulation of skeletal muscle. In the international literature the so-called compound method is recommended. The compound method includes the classical methods, algor and rigor and livor mortis, combined with additional methods like SMR [3-9]. SMR can be distinctive during the early post-mortem period when it comes to estimating the PMI, concerning cases in which classical do not provide a reliable estimation [10].

In the first hours after death the body temperature remains relatively stable, which is known as the post-mortem temperature plateau [1-11]. Next, the body temperature decreases in a sigmoidal shape until it reaches the ambient temperature. Body cooling is influenced by body characteristics such as body weight, clothing, underlying diseases, and medication or drugs. In addition, environmental factors like ambient temperature, and the specific circumstances at the place the person died can also influence the process of body cooling [1-3]. The rate of cell metabolism, i.e. the turnover of ATP and glycogene in muscle cells, is dependent on body temperature. SMR is, like rigor mortis, dependent on the storage of ATP and glycogene in muscle cells [1-4]. With higher body temperature a higher energy consumption is to be expected which could influence the outcome of SMR [12-15].

Warther et al. investigated SMR on 270 bodies in a mortuary of a hospital using a steel chisel, with a known PMI between 7- and 15-hours post-mortem. It concerned in- and out-of-hospital deaths of which some bodies were kept in a cold store for a varying duration of time before investigation. Warther et al. observed no significant influence of rectal temperature and surface temperature of the limbs on the outcome of SMR in the controlled environment of a mortuary [12].

The research of Warther et al. raises the question if the outcome of SMR is affected by rectal body temperature under uncontrolled environmental conditions, resembling daily forensic practice. In addition, compared to the musculus biceps brachii (MBB), the musculus brachioradialis (MBR) is located further away from the trunk of the body (Figure 1). The rate of body cooling is higher in more peripheral located muscles than in central, more proximal located muscles [16]. A lower body temperature leads to a lower rate of cell metabolism and subsequently to a prolonged availability of ATP. Therefore, we hypothesize that the outcome of SMR could differ between MBB and MBR on the same body examined at the same time.

Muscle excitability depends on endogenous factors like sex, age, and underlying diseases. On average, males have a higher skeletal muscle mass than females, while elderly people have a lower muscle mass than younger people due to muscle atrophy [17]. A higher muscle mass is associated with larger muscle fibres, based on a larger number of myofilaments and sarcomeres [17,18]. Therefore, it is conceivable that skeletal muscles in males store more ATP and glycogen compared to skeletal muscles in females. Accordingly, it is plausible that SMR can be observed for a longer period after death in males compared to females under the same contextual conditions.

Finally, antemortem, energy consuming underlying diseases such as infections or cancer, or a long period of sickness prior to death, could negatively influence the outcome of SMR [12,19].

In this study we focussed on the possible relationship between human body related, intrinsic variables, and post-mortem excitability of skeletal muscle. We investigated the influence of the body temperature, sex, age, state of rigor mortis, and the length of the period of sickness prior to death on the outcome of SMR. To test the posed hypotheses a field study was conducted during daily forensic medical practice, comprising of deceased in different environments.

Figure 1: Location of Mechanical Stimulation. 

a: musculus biceps brachii (MBB)

b: musculus brachioradialis (MBR)

A field study was conducted comprising of deceased in different environments. From January 2017 to August 2023, during regular external post-mortem investigation, four forensic physicians from two different regions in the Netherlands performed mechanical stimulation of both upper- and forearms of dead bodies by using a reflex hammer to trigger SMR [19]. The research population concerned in-hospital-deaths, out-of-hospital deaths that were transferred to the morgue of a hospital or a police mortuary for further investigation, and out-of-hospital deaths that were examined at the place where the body was found. In the case of out-of-hospital deaths it concerned deceased found on land in all kinds of indoor and outdoor settings, and submersed in water, such as in ditches, recreational lakes, rivers and swimming pools.

Inclusion criteria were adult (18 years and older) with a known or estimated PMI of less than 14 hours. The PMI was based on information derived from a medical file, police investigation or information derived from family members, acquaintances or neighbours of the deceased. The upper limit of 14 hours was based on the finding that until now SMR has been observed up to 14 hours post-mortem [4]. Exclusion criteria were severe trauma in terms of body destruction and bodies with signs of decomposition with a total decomposition score ≥ 7 on the Gelderman decomposition scale. In case of a score of ≥ 7 on the Gelderman decomposition scale at least one part of the corpse (face, torso, limbs) shows grey to green discoloration and/or skin blisters, skin slippage and/or marbling, and/or bloating [20].

The surfaces of the MBB and MBR were manually examined before mechanical stimulation to prevent a possible false positive result, due to an initially bumpy surface that could be interpreted as a positive SMR. Mechanical stimulation of the left and right musculus biceps brachii and the left and right proximal part of the musculus brachioradialis was achieved by a single blow with a reflex hammer. The outcome of SMR was considered positive (present) if a muscle contraction was detectable in at least one of the four stimulated muscles, either visibly or manually through palpation immediately after mechanical stimulation. If neither a visible nor palpable reaction was detected, the result was registered as negative (absent) [19].

The body temperature was measured with an industrial thermometer regularly used by forensic police investigators, expressing temperature in degrees Celsius. A body temperature in the range of 36.2 to 37.0 °C, measured rectally, was defined as normothermia of a body [21]. A body temperature lower than 36.2 °C was defined as hypotherm, a body temperature higher than 37.0 °C as hypertherm. The state of rigor mortis was manually and visually examined. The outcome was registered based on the stages described in the Dutch national register of forensic medicine (Formatus): absent, starting, maximum and decreasing, corresponding with the stadia mentioned in the current literature [22].

The length of the period of sickness prior to death was registered as follows: death less than 24 hours following the primary cause of death was registered as a ‘short period of sickness prior to death’; death longer than 24 hours following the primary cause of death was registered as a ‘long period of sickness prior to death’. The primary cause of death was defined as the initial event that induced a chain of events in the person’s body, which eventually led to the death of that person [23,24].

The Welch Two Sample t-test was performed to investigate if there was a significant difference in PMI, body temperature, and age between positive and negative SMR. The Welch Two Sample t-test assumes unequal variances and sample size of the data. The Chi-squared test was performed to investigate if there was a significant difference in the categorical variables sex and the length of the period of sickness prior to death between the outcomes of SMR. Both univariate and multivariate logistic regression were used to analyze how different variables influence the likelihood of SMR outcome. Univariate logistic regression was used to examine the individual effect of the state of rigor mortis and body temperature groups. Then, multivariate logistic regression was used to examine PMI, age, sex, length or the period of sickness prior to death, rigor mortis, and body temperature simultaneously to determine their combined effect on SMR. Statistical analyses were performed by using SPSS 27.0 and R 4.4.1. (https://www.R-project.org/). Significance was accepted at P<0.05.

In total 142 out of 364 cases with a known PMI were included, in which MBB and MBR on both arms were mechanically stimulated with a reflex hammer. It concerned 65 males (mean age: 73.4 ± 16.8 years) and 77 females (mean age: 82.1 ± 13.0 years). SMR was present in 74 out of 142 cases (52.1%). The mean PMI, mean rectal body temperature, and age did not differ significantly between cases with a positive SMR and cases showing no SMR (Table 1, P>0.05). The number of male cases that showed SMR (63.1%) was significantly higher than the number of female cases (42.9%) that showed SMR (Table 1, P<0.05). In the majority of the 142 cases (N=106, 74.6%) death occurred more than 24 hours after the incident (long period of sickness prior to death). There was no significant difference in outcome of SMR between persons who died less than 24 hours after the primary cause of death and persons who died more than 24 hours after the primary cause of death (Table 1, P>0.05).

Table 1: Outcome of SMR versus Mean PMI, Mean Age, Mean Rectal Body Temperature, Sex and Length of Period of Sickness Prior to Death (N=142).

*Acute = short period of sickness prior to death; less than 24 hours after primary cause of death), Chronic = long period of sickness prior to death; more than 24 hours after primary cause of death.

The percentage of a positive SMR decreased during successive stages of rigor mortis (Table 2). SMR could not be triggered in the 5 cases which showed a maximum state of rigor mortis. The stage of rigor mortis does not significantly determine the outcome of SMR (Table 3, P>0.05).

Table 2: The Observed Outcome of SMR versus Rigor Mortis (N = 142).

Table 3: Univariate Logistic Regression for Rigor Mortis as Predictor for the SMR Outcome (Dependent Variable: SMR Present, N=142).

* (Intercept) = rigor mortis absent.

In 76 out of the 142 cases (53.5%) the rectal body temperature at the moment of investigation was higher than 37.0 °C. SMR was present in 40 out of 76 cases (52.6%). In the other 66 cases it concerned bodies with normothermia (36.2 – 37.0 °C) or cooled bodies (< 36.2 °C) at the moment of muscle stimulation. In 34 out of the 66 cases (51.5%) SMR was present (figure 2). Being in one of the three clusters of rectal body temperature does not significantly determine the outcome of SMR (Table 4, P>0.05).

Figure 2: Body Temperature versus SMR (N = 142).

*hypothermia < 36.2 °C, normothermia: 36.2 – 37.0 °C, hyperthermia > 37.0 °C

Table 4: Univariate Logistic Regression for Cluster Temperatures as Predictor for the SMR Outcome (Dependent Variable: SMR Present, N=142).

*(Intercept) = normothermia group.

In daily practice the forensic physician must deal with the presence of all the intrinsic variables analysed in this study while investigating a body, which implies a possible mutual influence on the outcome of SMR. Therefore, use of a holistic model combining all these investigated variables is more suited to daily forensic work. Application of this holistic model confirms that significantly more males show a positive SMR compared to females (Table 5, P < 0.05), while no relation was observed between age, rectal body temperature, rigor mortis and the length of period of sickness prior to death, and the outcome of SMR (Table 5, P>0.05). Thus, application of the holistic model confirms that only sex must be taken into account concerning the outcome of SMR in the early hour’s post-mortem.

Table 5: Multivariate Logistic Regression for Sex, Age, Length of Period of Sickness Prior To Death, Rectal Body Temperature, and Rigor Mortis as Predictors for the SMR Outcome (Dependent Variable: SMR Present, N=142).

*(Intercept): sex = female, acute (Short period of sickness prior to death; less than 24 hours after primary cause of death), age/body temperature/rigor mortis/PMImin = 0

** Chronic: Long period of sickness prior to death; more than 24 hours after primary cause of death

In 114 out of the 142 cases (80.3%) the outcome of SMR was the same for both musculus biceps brachii (MBB) and musculus brachioradialis (MBR) on both arms; in 46 cases SMR was present and in 68 cases SMR was absent. In 9 cases SMR was present after stimulation of the MBB while absent after stimulation of the MBR on the same body. In 19 cases SMR was present after stimulation of the MBR while absent after stimulation of the MBB on the same body.

In 28 out of 142 cases (19.7%) that showed SMR, there was a discrepancy in the outcome between the MBB and the MBR. Comparing the 9 cases showing only a reaction of MMB with the 19 cases showing only a reaction of MBR, no significant relationships were observed concerning rectal body temperature, sex, age, PMI, rigor mortis and the period of sickness prior to death (Table 6, P>0.05).

Table 6: Multivariate Logistic Regression For Sex, Age, Length Of Period Of Sickness Prior To Death, Rectal Body Temperature, And Rigor Mortis As Predictors For The Outcome Of Either MBB Or MBR (Dependent Variable: MBB, N=28).

*(Intercept): sex = female, acute (Short period of sickness prior to death; less than 24 hours after primary cause of death), normothermia group, age/rigor mortis = 0

** Chronic: Long period of sickness prior to death; more than 24 hours after primary cause of death

Compared to the study of Warther et al., in this study a substantial higher number of cases (52.1%) showed a positive SMR, which can be explained by a shorter PMI compared to the cases included in the study of Warther et al [12]. However, in 47.9% of cases SMR was not observed despite the short PMI, suggesting that other factors influence the outcome of SMR in the first hours after death.

The percentage of male cases that showed SMR was significantly higher compared to the percentage of female cases that showed SMR in the early hour’s post-mortem. This significant difference in the outcome of SMR between males and females for the early hours post-mortem period suggests a possible influence of specific sex related aspects that already can be observed in case of a short PMI. This observed difference could be due to the earlier mentioned physiological differences between males and females, such as differences in post-mortem energy supply in muscle cells and the capacity of male skeletal muscle to generate more power on a general basis. Hence, with longer PMI’s these physiological differences could become more evident subsequently leading to observable differences in outcome of SMR. Therefore, in future studies, it is necessary to have larger sample sizes, a wider range of PMIs, such as the range used by Warther et al., to establish if there is a difference between males and females regarding the upper limit of PMI in which SMR can be observed.

Since the 19th century it is known that the body temperature of a human being does not drop immediately after death [11]. We suggested that a higher body temperature could influence the outcome of SMR in uncontrolled environmental circumstances, due to a higher energy consumption in muscle cells. In a majority of cases (53.5%) the bodies were hyperthermic at the moment of muscle stimulation, which logically implicates hyperthermia peri-mortem. In the current study, for the early hour’s post-mortem, the temperature of the body at the time of stimulation had no significant effect on the outcome of SMR. However, it remains unclear, based on the short PMI of the cases included in this study, whether there is a relationship between the body temperature and the outcome of SMR in case of a higher PMI, i.e. a PMI ranging from 5 to 13 hours. 

The percentage of positive outcomes of SMR decreased during successive stages of rigor mortis for both MBB and MBR. As expected, this result coincides with an increasing mean PMI, which is associated with a decreasing energy supply in muscle cells. No SMR was observed on bodies with fully developed rigor mortis. This suggests that from the moment rigor mortis is fully developed, SMR has no added value. However, the univariate logistic regression showed that the state of rigor mortis does not significantly determine the outcome of SMR, which could be due to a small sample size. Therefore, to substantiate the assumption that SMR has no added value in cases with fully developed rigor mortis, a larger study population of bodies with fully developed rigor mortis is necessary.

We hypothesized that the period of sickness prior to death, could influence the outcome of SMR after mechanical stimulation of skeletal muscle [12]. While SMR is dependent on post-mortem energy supply in muscle cells, less SMR is to be expected in case of a long period of sickness prior to death. However, in the current study, in 51 out of 105 cases (48.6%) who died after 24 hours after the primary cause of death, SMR could be observed. Compared to the 37 cases in which the subject died within 24 hours after the primary cause of death, of which 22 cases showed SMR (59.5%), there was no significant difference in the outcome of SMR between both subgroups. The majority of the included study population was examined within 400 minutes (6.67 hours) after death. The shorter the PMI, the more energy supply in muscle cells is to be expected, hence a higher likelihood of a positive SMR outcome. Thus, the shorter PMI in this study for the cases with a long period of sickness prior to death could have made a positive outcome of SMR more likely. So, it seems the period of sickness prior to death does not influence the outcome of SMR in the early hours after death.

A possible explanation for the observed difference in outcome of SMR between MBB and MBR of the same body could be variation in the force applied to the muscle [25].  In daily forensic practice, it is unavoidable that there is intra- and interpersonal variation in the applied force by forensic physicians. The MBR is located more superficial compared to the MBB. On average, between the skin and the MBB there is more subcutaneous tissue compared to the MBR. So, the same magnitude of force applied to the arm possibly could absorb more of the energy generated at the location where the skin is hit in case of the MBB compared to the MBR. Subsequently, less energy could reach the surface of MBB compared to the MBR, which could have had a negative influence on the outcome of SMR. Thus, it is recommended to investigate both muscles on both arms concerning SMR to avoid false negative outcome of SMR.

The current study shows a significant difference in outcome of SMR between male and female deceased in the early hours after death. In addition, rectal body temperature, age, rigor mortis and the period of sickness prior to death did not significantly influence the outcome of SMR.

A discrepancy in the outcome of SMR was observed between the MBB and the MBR on the same body in 19.7% of cases, which could be due to local tissue characteristics, such as the influence of adipose tissue, or differences in the applied force of the mechanical stimulation. Therefore, to provoke SMR, mechanical stimulation of MBB and MBR of both arms is recommended to minimalize a false negative outcome of SMR. Also, provoking SMR by mechanical stimulation should be performed regardless of the state of rigor mortis.

Compliance with Judicial and Ethical Standards

The post-mortem investigation of the bodies was conducted according to standard forensic procedures. No additional actions were carried out. 

Research Involving Human Participants And/Or Animals

In accordance with the Central Committee on Research Involving Human Subjects (https://www.ccmo.nl), the Medical Research Involving Human Subjects Act (https://wetten.overheid.nl/BWBR0009408/2022-01-31) does not apply for the data collected in this study (METC-number 2023-0269).

Conflict of Interest Statement

The authors declare that they have no conflict of interest.

Funding

None.

Author Contribution Declaration

Stigter H, Duijst WLJM collected data in the field. Stigter H, Krap T and Kloosterman J analyzed the data. Stigter H, Duijst WLJM, Krap T and Kloosterman J wrote the manuscript. Duijst WLJM and Krap T supervised the project.

Acknowledgement

We sincerely would like to express our appreciation to Ms. L. Dijkhuizen and Ms. T. Gelderman M.D., for their contributions in collecting the data used for this study, and Ms. A. Gautier for providing the drawing used in Figure 1.

Human Ethics and Consent to Participate Declarations

Not applicable. 

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