We Check the Time of Death with Post-Mortem Degradation of Skeletal Proteins – And Other Gruesome Forensic Facts of Life

A photomontage showing a body outline, "Do Not Cross" yellow police tape, and a magnifier focused on the word "Forensics". The picture also lists the following forensic ways to estimate the time of death - Wait: Determine the time of death; Rigor mortis; Decomposition; Forensic Entomology; Succession on corpses; Other decomposers.Estimating the Time of Death

Forensic researchers from the University of Salzburg have developed a new method for establishing an exact time of death after as long as 10 days – a significant step forward from the current method of measuring core body temperature, which only works up to 36 hours after death.

When a human body is discovered, one of the key questions is ‘When did the person die?’ – the answer to which is critical in cases of suspicious death.  The accuracy of estimating the time since death is negatively correlated with the time elapsed since death occurred.

The main early post-mortem changes to the human body include:

  • changes in temperature

  • hypostasis

  • rigor mortis

  • changes in the eyes.

In Forensic Science, the time since death is known as the ‘post-mortem interval‘ (PMI).  After the first few days, and up to several months after death, entomology – the science of insects and invertebrates present on or around the decomposing body – may also yield valuable information about the post-mortem interval.

Estimating the time since death is a very important aspect in forensic sciences which has been pursued by a variety of methods. 


Our Body Temperature

During life…  “Thank goodness”, you think!  “We’re talking about life again…  All that forensic talk was getting really dreary…”  Bear with…  It IS science!! 

A graph showing the natural rate of post-mortem cooling, according to Newton's Law of Cooling Postmortal Temperature.
The natural human body post-mortem cooling curve follows Newton’s Law of Cooling  Source: forensicmed.co.uk

During life, then, the core temperature of human beings is normally maintained around a constant value of 37°C.  After death, the mechanisms that regulate this steady temperature cease and the body ultimately attains the temperature of its environment.  Thus, the body cools in those parts of the World where ambient temperature is below 37°C.

The drop in body temperature is approximately exponential.  Newton’s Law of Cooling states that the rate of heat loss of a body is proportional to the difference in temperatures between the body and its surroundings.

If the temperature of the environment ranges from 16-20°C, the core body temperature at 6, 10 and 15 hours after death, may be 30-34°C, 28°C and 24-26°C respectively.

Importantly, we need realise that many factors can affect the rate of cooling of a corpse, including:

  • atmospheric conditions, i.e. air temperature, humidity, precipitation

  • initial body temperature

  • amount of clothes worn by victim

  • body weight to surface area ratio of the deceased.

After about 42 hours, when the process of decomposition is well on its way, the body core temperature is expected to rise slightly.


Early Post-Mortem Changes

This photograph shows the bottom half of a deceased person's face, illustrating the dark discolouration of the drying lips, known as Livor Mortis.
Livor Mortis: A dark discoloration and drying of the lips is common following death. Source: DocumentingReality.com

Hypostasis or Livor Mortis develops a short time after death.  Post-mortem lividity becomes apparent within one hour, fully developed after 3-4 hours, and persists until decomposition.

Normally, hypostasis is apparent as a dark pink or dark purple-blue discoloration, similar in appearance to bruising, caused by the settlement of blood in the lowermost parts.

When the normal blood circulation has ceased at the point of death, hypostasis gradually sets in.  Under the influence of gravity, the blood passively flows and settles in the blood vessels of the lowest parts of the body.

A photograph showing the mid-part of a deceased person's body, illustrating the phenomenon of hypostasis or livor mortis.
Post-Mortem Hypostasis (Livor Mortis) Source: forensicmed.co.uk

It does not occur in areas of the body where pressure is exerted.  The blood flow into the capillaries is prevented and the areas affected remain characteristically pale.

In poisoning deaths, the colouring of the post-mortem lividity is markedly different.  Notably, a cherry-pink colouration indicates carbon monoxide CO poisoning.

The main use of hypostasis is to ascertain whether or not the original position or location of a body has been changed since death.  Within a few hours of death, the original areas of lividity tend to disappear and re-form in new areas, consistent with the new position of the body, if it has been moved.  If these are inconsistent, with the position of the body when discovered, it strongly indicates that the body has been moved from its original position several hours following death.

Although it can provide some insight if the death is comparatively recent, this phenomenon is a far less reliable indicator of time of death than rigor mortis.

A photograph showing a deceased's hand clutching at vegetation, illustrating the phenomenon of rigor mortis.
Rigor Mortis: Occasionally, rigor mortis occurs very rapidly, as in this drowning victim, still clutching at vegetation, in a so-called cadaveric spasm. Source: DocumentingReality.com

Rigor mortis is the characteristic stiffening of the body that occurs in the early stages after death.  Brought on by complex post-mortem biochemical changes, rigor mortis is caused by temporary rigidity within both voluntary and involuntary muscle systems. 

Following death, the body is flaccid.

Within 4-6 hours, rigor mortis sets in, and develops normally in a standard fashion, starting with the facial muscles, the jaw and neck muscles, extending downward to the arms, trunk and legs.

Within 12-18 hours, the entire body is rigid.

From 24-36 hours after death occurred, rigor mortis then gradually disappears in the same order as it appeared.  The body is then flaccid once again.

Rigor mortis provides information relative to any change of location of the body following death, all the more apparent if a rigid body happens to be inconsistent with its surroundings.

However, estimating the time of death with rigor mortis is a highly unreliable technique, as the whole process is influenced by environmental temperatures.  In hot climates, rigor mortis starts early, progresses quickly and disappears rapidly compared to cooler climes.

Additionally, the intensity of the rigidity varies with each individual’s level of muscular development.


Changes in the Eyes

A photograph showing the postmortem change in the eye of a deceased, illustrating the "Tâche Noire de la Sclérotique".
Tâche Noire de la Sclérotique is one of the ocular signs of death in which a reddish brown discoloration is transversely arranged across the sclera. It occurs when the eyes are not completely closed so that the sclera is exposed to air. Source: emedicine.Medscape.com

The eyes of the deceased can also be used to estimate the time since death.  Early post-mortem changes that occur in the eyes of the deceased:

  • potassium in vitreous humour of the eye
  • cloudiness.

Potassium is slowly released into the clear gelatinous material that makes up the bulk of the eyeball behind the lens, from the post-mortem decay of the blood cells of the retina.  

The rate of potassium release can be used to calculate the PMI.

The potassium release rate can be used to calculate the PMI.


During life, the crystalline structure of the cornea and the lens is dependent on hydration.  After death, the water of crystallisation is lost and the eye desiccates and becomes opaque.

Once again, these changes are affected by environmental factors whether or not the eyes are shut.

Overall, it seems body temperature represents the best measure available for estimating the time of death by back-calculation, especially within the first 18 hours after death.

Until now…


A Novel Approach – Postmortem Degradation of Skeletal Proteins…

Several slides showing stem cells in skeletal muscles post mortem. The study compares: a) Control human muscle b) Human muscle 17 days post-mortem c) Human muscle in an organ donor 30 days post-section d) Mouse cadaver muscle 8 days post mortem.
Skeletal muscle stem cells survive for extended periods post mortem. Source: Chrétien et al., 2012 Nature.com

There have been a shortage of “reliable” methods to calculate the time of death after the moment when the body has cooled down to environmental temperatures, which depending on the ambient temperature can normally take about one to two days.

The most precise method to determine the post-mortem interval consists in the temperature method, based on the decrease of the body core temperature from 37 °C, as outlined above.  However, this method is only useful in the early postmortem phase (~ 0-36 hours).

Forensic scientists have long been searching for a new way to assess the time of death after this.  The aim of the new study was to develop an accurate method for PMI determination beyond this present limit…

The breakthrough was announced at the Society for Experimental Biology‘s annual conference in Prague.  The team from Austria’s University of Salzburg measured the breakdown of muscle proteins in dead pigs over time.  They studied the muscle proteins of pigs, because of their close similarity to human muscles.

The protein building blocks of our muscles are very large, tangled molecules that, after death, begin to break down into smaller pieces.  For some of the proteins, this happens in a very specific time frame.  Even the breakdown products are present for a specific time.

The breakdown products are present for a specific time.  So if you know which of these products are present, you know exactly when the individual died.

A composite photograph with chemical molecular models superimposed on a picture of beakers and pipette involved in chemical manipulation.
Checking the Time of Death – A Question of Chemistry? Post Mortem Degradation of Skeletal Proteins  Source: Forensic News.com

The aim of the study was to develop an accurate method for PMI determination beyond the present limit.  For this purpose, they used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and casein zymography to analyse the time course of degradation of selected proteins and calpain activity in porcine biceps femoris muscle until 240 hours post-mortem.

Their results demonstrated that titin, nebulin, desmin, cardiac troponin T, and SERCA1 degraded in a regular and predictable fashion in all samples investigated.  Similarly, both the native calpain 1 and calpain 2 bands disintegrate into two bands subsequently.

This degradation behaviour identifies muscular proteins and enzymes as promising substrates for future molecular-based PMI determination technologies.

If you know which of these products are present in a sample, then you know when the individual died.


The team also analysed more than 60 human tissue samples from the forensic department of the same university. And their preliminary findings showed similar clockwork-like changes.  They now need more samples to find out whether gender, body mass index, temperature, humidity, may have an impact on the time-course of muscle breakdown.

Any research that could assist in helping narrow down a time of death is always of value.  But it will take some time before this is validated for court use.  Forensic and legal community will need to be very convinced that there are no confounding factors, before we come to rely on this evidence to convict someone.

The scientists found this muscle protein degradation proved to be a very promising method.  Within three years, this technique could help in the gathering of vital forensic evidence.